Combination of nanoindentation and quantitative backscattered electron imaging revealed altered bone material properties associated with femoral neck fragility.

Osteoporotic fragility fractures were hypothesized to be related to changes in bone material properties and not solely to reduction in bone mass. We studied cortical bone from the superior and inferior sectors of whole femoral neck sections from five female osteoporotic hip fracture cases (74-92 years) and five nonfractured controls (75-88 years). The typical calcium content (Ca(Peak)) and the mineral particle thickness parameter (T) were mapped in large areas of the superior and inferior regions using quantitative backscattered electron imaging (qBEI) and scanning small-angle X-ray scattering, respectively. Additionally, indentation modulus (E) and hardness (H) (determined by nanoindentation) were compared at the local level to the mineral content (Ca(Ind)) at the indent positions (obtained from qBEI). Ca(Peak) (-2.2%, P = 0.002), Ca(Ind) (-1.8%, P = 0.048), E (-5.6%, P = 0.040), and H (-6.0%, P = 0.016) were significantly lower for the superior compared to the inferior region. Interestingly, Ca(Peak) as well as Ca(Ind) were also lower (-2.6%, P = 0.006, and -3.7%, P = 0.002, respectively) in fracture cases compared to controls, while E and H did not show any significant reduction. T values were in the normal range, independent of region (P = 0.181) or fracture status (P = 0.551). In conclusion, it appears that the observed femoral neck fragility is associated with a reduced mineral content, which was not accompanied by a reduction in stiffness and hardness of the bone material. This pilot study suggests that a stiffening process in the organic matrix component contributes to bone fragility independently of mineral content.

The three-dimensional arrangement of the mineralized collagen fibers in elephant ivory and its relation to mechanical and optical properties.

Elephant tusks are composed of dentin or ivory, a hierarchical and composite biological material made of mineralized collagen fibers (MCF). The specific arrangement of the MCF is believed to be responsible for the optical and mechanical properties of the tusks. Especially the MCF organization likely contributes to the formation of the bright and dark checkerboard pattern observed on polished sections of tusks (Schreger pattern). Yet, the precise structural origin of this optical motif is still controversial. We hereby address this issue using complementary analytical methods (small and wide angle X-ray scattering, cross-polarized light microscopy and scanning electron microscopy) on elephant ivory samples and show that MCF orientation in ivory varies from the outer to the inner part of the tusk. An external cohesive layer of MCF with fiber direction perpendicular to the tusk axis wraps the mid-dentin region, where the MCF are oriented mainly along the tusk axis and arranged in a plywood-like structure with fiber orientations oscillating in a narrow angular range. This particular oscillating-plywood structure of the MCF and the birefringent properties of the collagen fibers, likely contribute to the emergence of the Schreger pattern, one of the most intriguing macroscopic optical patterns observed in mineralized tissues and of great importance for authentication issues in archeology and forensic sciences. STATEMENT OF SIGNIFICANCE: Elephant tusks are intriguing biological materials as they are composed of dentin (ivory) like teeth but have mineralized collagen fibers (MCF) similarly arranged to the ones of lamellar bones and function as bones or antlers. Here, we showed that ivory has a graded structure with varying MCF orientations and that MCF of the mid-dentin are arranged in plywood like layers with fiber orientations oscillating in a narrow angular range around the tusk axis. This organization of the MCF may contribute to ivory's mechanical properties and, together with the collagen fibers birefringence properties, strongly relates to its optical properties, i.e. the emergence of a macroscopic checkerboard pattern, well known as the Schreger pattern.

Ultrafine heat-induced structural perturbations of bone mineral at the individual nanocrystal level.

The nanoscale characteristics of the mineral phase in bone tissue such as nanocrystal size, organization, structure and composition have been identified as potential markers of bone quality. However, such characterization remains challenging since it requires combining structural analysis and imaging modalities with nanoscale precision. In this paper, we report the first application of automated crystal orientation mapping using transmission electron microscopy (ACOM-TEM) to the structural analysis of bone mineral at the individual nanocrystal level. By controlling the nanocrystal growth of a cortical bovine bone model artificially heated up to 1000 °C, we highlight the potential of this technique. We thus show that the combination of sample mapping by scanning and the crystallographic information derived from the collected electron diffraction patterns provides a more rigorous analysis of the mineral nanostructure than standard TEM. In particular, we demonstrate that nanocrystal orientation maps yield valuable information for dimensional analysis. Furthermore, we show that ACOM-TEM has sufficient sensitivity to distinguish between phases with close crystal structures and we address unresolved questions regarding the existence of a hexagonal to monoclinic phase transition induced by heating. This first study therefore opens new perspectives in bone characterization at the nanoscale, a daunting challenge in the biomedical and archaeological fields, which could also prove particularly useful to study the mineral characteristics of tissue grown at the interface with biomaterials implants. STATEMENT OF SIGNIFICANCE: In this paper, we propose a new approach to assess the mineral properties of bone at the individual nanocrystal level, a major challenge for decades. We use a modified Transmission Electron Microscopy acquisition mode to perform an Automated Crystal Orientation Mapping (ACOM-TEM) by analyzing electron diffraction patterns. We tune the mineral nanocrystal size by heating a model bovine bone system and show that this method allows precisely assessing the mineral nanocrystal size, orientation and crystallographic phase. ACOM-TEM therefore has sufficient sensitivity to solve problems that couldn't be answered using X-ray diffraction. We thus revisit the fine mechanisms of bone nanocrystal growth upon heating, a process currently used for bone graft manufacturing, also of practical interest for forensic science and archaeology.

Extracellular and intracellular fluid volume monitoring during dialysis by multifrequency impedancemetry.

Impedance techniques can, in principle, permit non-invasive monitoring of extracellular (Ve) and intracellular (Vi) fluid volumes during dialysis. The authors present a method that determines the resistances Re and Ri of extracellular and intracellular compartments, respectively, by extrapolating impedance measurement toward zero, as well as infinite frequencies, according to the Cole-Cole model of biologic issues. These measurements were made using a XITRON 4000 B impedance meter (Xitron Technologies Inc., San Diego, CA) at frequencies ranging from 5 kHz to 1 MHz. The fact that the body is not a cylindric, homogeneous conductor is taken into account by introducing shape factors ke and ki and different resistivities pe and pi for the extracellular and intracellular fluid compartments. Assuming that these four unknown parameters can be regarded as constant during dialysis, the authors obtain: [formula: see text] where subscript o denotes initial values at start of dialysis. Impedances were measured at 30 min intervals on 11 pediatric patients and two adults, whereas total body water volume was determined by measuring urea in collected dialysate. Without ultrafiltration, Ve and Vi do not change significantly in percentage whereas, in the presence of ultrafiltration, Ve decreased by 15% to 25%. In cases when Vi does not change, it is possible to determine Ve and its variation during dialysis.

Extra- and intracellular volume monitoring by impedance during haemodialysis using Cole-Cole extrapolation.

A method is presented for monitoring the relative variation of extracellular and intracellular fluid volumes using a multifrequency impedance meter and the Cole-Cole extrapolation technique. It is found that this extrapolation is necessary to obtain reliable data for the resistance of the intracellular fluid. The extracellular and intracellular resistances can be approached using frequencies of, respectively, 5 kHz and 1000 kHz, but the use of 100 kHz leads to unacceptable errors. In the conventional treatment the overall relative variation of intracellular resistance is found to be relatively small.

Spiral twisting of fiber orientation inside bone lamellae.

The secondary osteon - a fundamental building block in compact bone - is a multilayered cylindrical structure of mineralized collagen fibrils arranged around a blood vessel. Functionally, the osteon must be adapted to the in vivo mechanical stresses in bone at the level of its microstructure. However, questions remain about the precise mechanism by which this is achieved. By application of scanning x-ray diffraction with a micron-sized synchrotron beam, along with measurements of local mineral crystallographic axis direction, we reconstruct the three-dimensional orientation of the mineralized fibrils within a single osteon lamella ( approximately 5 mum). We find that the mineralized collagen fibrils spiral around the central axis with varying degrees of tilt, which would - structurally - impart high extensibility to the osteon. As a consequence, strains inside the osteon would have to be taken up by means of shear between the fibrils.

Recent synchrotron radiation microdiffraction experiments on polymer and biopolymer fibers.

The status of synchrotron radiation (SR) microdiffraction techniques developed at the ID13 beamline of the European Synchrotron Radiation Facility (ESRF) is reviewed for polymer and biopolymer fiber applications. Beam sizes in the micrometer-range have been used to study the local structure of whole fibers such as viscose-rayon or poly(p-phenylene terephthalamide). The possibilities for in situ studies during stretching, extrusion, or indentation will be discussed.