Bright Lamellae Research Articles

Lamellar thickness measurements in control and osteogenesis imperfecta human bone, with development of a method of automated thickness averaging to simplify quantitation

Lamellar bone that forms in moderate and severe osteogenesis imperfecta (OI) is composed of structurally irregular lamellae compared to those in control bone. OI and control cortical bone fragments were prepared for light microscopy in standardized fashion: decalcified, embedded in plastic, sectioned and stained with toluidine blue. Polarization light microscopy (PLM) was used to demonstrate and quantify bright and dark lamellar thicknesses in cortical bone fragments from 5 patients with moderate to severe OI in whom type I collagen structural/molecular defects were detected and in control bone from 5 patients. Rigid selection criteria identified lamellar regions for quantification. Thicknesses of bright and dark lamellae were measured manually at 20X magnification using a histomorphometric image analysis system. A method of automated thickness averaging was developed to determine lamellar thicknesses from PLM images to make measurement faster. Our study demonstrates, for the first time, that in OI bone from patients with type I collagen structural/molecular defects mean lamellar thickness measurements (along with the bright and dark lamellar thicknesses) were less than those in control bone by statistically highly significant differences. The mean value for bright lamellae was less than that for dark lamellae in both control and OI bone. The ratio of mean values for bright/dark lamellar thicknesses was the same in control and OI bone. The automated method obtained similar results to the manual method. Lamellar bone in moderate and severe OI with type I collagen defects is composed of thinner and less structurally regular lamellae than those in control bone. This finding indicates that lamellar thickness measurements can be helpful in assessing the effect of specific collagen and collagen-related mutations on OI bone synthesis and warrant inclusion in research and clinical histomorphometric assessments.

Effect of cortical bone micro-structure in fragility fracture patients on lamellar stress

This work investigates how changes in cortical bone microstructure alter the risk of fragility fractures. The secondary osteons of non-osteoporotic (by DXA) women with fragility fractures have reduced lamellar width and greater areas of birefringent brightness in transverse sections, a pathological condition. We used hierarchical finite element (FE) models of the proximal femur of two women aged 67 and 88 (younger and older) during one-legged stance. At specific locations of the anterior-inferior neck (ROI), we analyzed micro-models containing osteons comprised of alternating birefringent extinct and bright lamellae. The plane of lamellar isotropy (XY) was transverse to the osteon longitudinal axis (Z) which was parallel to the femoral neck axis. To evaluate changes in fracture risk with changes in microstructure, we investigated principal and von Mises stresses, and planar stress measures that accounted for transverse isotropy. For both younger and older femurs, 48% to 100% of stress measures were larger in models with healthy architecture than in models with pathological architecture, while controlling for type of lamella and osteon. These findings suggest that bone adaptation reduces stress at most pathological lamellar sites. However, in the bright lamellae of the younger femur, the pathological tensile, compressive and distortional stresses in the transverse plane and distortional stress in the longitudinal planes were larger than the non-negligible corresponding stresses in 6 of the 28 osteon models with healthy architecture, in 5 of the 7 locations. Therefore, a minority of sites with pathological architecture present greater stress, and therefore, greater fracture risk.

Parathyroid hormone treatment improves the cortical bone microstructure by improving the distribution of type I collagen in postmenopausal women with osteoporosis.

Although an important index, the level of bone mineral density (BMD) does not completely describe fracture risk. Another bone structural parameter, the orientation of type I collagen, is known to add to risk determination, independently of BMD, ex vivo. We investigated the Haversian system of transiliac crest biopsies from postmenopausal women before and after treatment with parathyroid hormone (PTH). We used the birefringent signal of circularly polarized light and its underlying collagen arrangements by confocal and electron microscopy, in conjunction with the degree of calcification by high-resolution micro-X-ray. We found that PTH treatment increased the Haversian system area by 11.92 ± 5.82 mm² to 12.76 ± 4.50 mm² (p = 0.04); decreased bright birefringence from 0.45 ± 0.02 to 0.40 ± 0.01 (scale zero to one, p = 0.0005); increased the average percent area of osteons with alternating birefringence from 48.15% ± 10.27% to 66.33% ± 7.73% (p = 0.034); and nonsignificantly decreased the average percent area of semihomogeneous birefringent osteons (8.36% ± 10.63% versus 5.41% ± 9.13%, p = 0.40) and of birefringent bright osteons (4.14% ± 8.90% versus 2.08% ± 3.36%, p = 0.10). Further, lamellar thickness significantly increased from 3.78 ± 0.11 µm to 4.47 ± 0.14 µm (p = 0.0002) for bright lamellae, and from 3.32 ± 0.12 µm to 3.70 ± 0.12 µm (p = 0.045) for extinct lamellae. This increased lamellar thickness altered the distribution of birefringence and therefore the distribution of collagen orientation in the tissue. With PTH treatment, a higher percent area of osteons at the initial degree of calcification was observed, relative to the intermediate-low degree of calcification (57.16% ± 3.08% versus 32.90% ± 3.69%, p = 0.04), with percentage of alternating osteons at initial stages of calcification increasing from 19.75 ± 1.22 to 80.13 ± 6.47, p = 0.001. In conclusion, PTH treatment increases heterogeneity of collagen orientation, a starting point from which to study the reduction in fracture risk when PTH is used to treat osteoporosis.

Microstructural investigations on strongly stained olivines of the chassignite NWA 2737 and implications for its shock history

Olivines from the meteorite Northwest Africa 2737 (NWA 2737) show unique characteristics such as strong brownish staining, the occurrence of perpendicular sets of bright lamellae and the exsolution of metallic nanoparticles. These features were investigated by transmission electron microscopy and spectroscopic techniques to understand their formation and consequent implications for the shock history of NWA 2737. Areas showing optically an intense brown staining are characterised by the occurrence of finely dispersed metallic nanoparticles, an extreme high density (10 16 m − 2 ) of polygonised dislocations with Burgers vector [001] and a high abundance of trivalent iron (16%) as determined by electron energy loss spectroscopy. In contrast, the bright lamellae contain individual dislocation lines with Burgers vector [001] of a slightly lower density (10 14 m − 2 ), almost no metallic nanoparticles and significantly lower trivalent iron content. Model mass balance calculations suggest that the high content of trivalent iron can be incorporated as a laihunite component into the cores of polygonised dislocations. This is charge balanced by the formation of metallic nanoparticles. Trivalent iron is likely responsible for the intense brown colour, whereas nanoparticles darken these areas considerably and cause an intense red slope in the infrared range. The proposed diffusion of trivalent iron into the cores of polygonised dislocations and the associated exsolution of metallic iron nanoparticles are likely fundamental processes for intense olivine staining during meteoritic impacts in general. Detailed analyses of variously stained regions suggest that the bright lamellae have experienced slightly higher shock-induced strain causing a recrystallisation of olivine, which occurred simultaneously with the polygonisation and exsolution processes resulting in the olivine staining of adjacent areas. The existence of individual dislocations can be interpreted as the result of a two-stage shock history for NWA 2737. After a first impact, the material of NWA 2737 was likely embedded in a hot ejecta blanket. This would ensure sufficient time at elevated temperatures for the polygonisation of dislocations and the recrystallisation of bright lamellae. A second impact resulted in the emission of individual dislocations into recrystallised olivine and the ejection of that meteorite from Mars.

Orientation of collagen at the osteocyte lacunae in human secondary osteons

This work characterizes an aspect of human bone micro-structure, pertinent to fracture initiation and arrest. It addresses how the orientation of elementary components proximate to osteocyte lacunae influences secondary osteon micro-biomechanics. New data at the perilacunar region concerning orientation of collagen-apatite, and prior data on collagen orientation outside the perilacunar region, are incorporated in a novel simulation of osteons to investigate how orientation relates to strains and stresses during mechanical testing. The perilacunar region was observed by confocal microscopy within single lamellar specimens, isolated from osteons. The specimens were separated by extinct or bright appearance in transverse section under circularly polarizing light. This is because synchrotron diffraction and confocal microscopy had established that each type, away from the perilacunar region, corresponds to specific dominant collagen orientation (extinct lamellae's dominant collagen forming small angles with the original osteon axis, while the bright lamellae's forms larger angles). Morphometry of serial confocal images of each perilacunar region showed collagen orientation generally following the orientation of canaliculi, circumambiently-perpendicular to the lacuna. The lacunae tilted relative to the lamellar walls were more numerous in extinct than in bright lamella. Their apices were less likely in extinct than bright lamella to show collagen following the canalicular orientation. The simulation of osteocyte lacunae in osteons, under tension or compression loading, supports the hypothesis that collagen orientation affects strains and stresses at the equatorial perilacunar region in conjunction with the presence of the lacuna. We further conjecture that collagen orientation diverts propagation of micro-cracks initiating from apices.

Collagen orientation patterns in human secondary osteons, quantified in the radial direction by confocal microscopy

The composite structure of secondary osteon lamellae, key micro-mechanical components of human bone, has intrigued researchers for the last 300 years. Scanning confocal microscopy here for the first time systematically quantifies collagen orientations by location within the lamellar thickness. Fully calcified lamellar specimens, extinct or bright in cross-section under circularly polarized light, were gently flattened, and then examined along their thickness direction, the radial direction in the previously embedding osteon. Collagen orientation was measured from confocal image stacks. So-called extinct lamellae and so-called bright lamellae are found to display distinct, characteristic patterns of collagen orientation distribution. Orientations longitudinal to the osteon axis in extinct lamellae, transverse to the osteon axis in bright lamellae, and oblique to the osteon axis in both lamellar types, show parabolic distribution through specimen thickness. Longitudinal collagen in extinct lamellae, and transverse collagen in bright lamellae, peaks at middle third of lamellar thickness, while oblique collagen peaks at outer thirds of both types. Throughout the thickness, longitudinal collagen orientations characterize extinct lamellar specimens, while orientations oblique to the original osteon axis characterize bright lamellar specimens. Measured patterns complement previous indirect results by different methods and reinforce previously hypothesized differences in lamellar mechanical functions.

Structural differences between “dark” and “bright” isolated human osteonic lamellae

This investigation presents new insights into the structure of human secondary lamellae. Lamellar specimens that appear dark and bright on alternate osteon transverse sections under circularly polarizing light were isolated using a new technique, and examined by polarizing light microscopy, synchrotron X-ray diffraction, and confocal microscopy. A distribution of unidirectional collagen bundles and of two overlapping oblique bundles appears on circularly polarizing light microscopy images in relation to the angle between the specimen and the crossed Nicols’ planes. The unidirectional collagen bundles observed at 45° run parallel to the osteon axis in the dark lamellar specimens and perpendicular to it in the bright ones. Small and wide-angle micro-focus X-ray diffraction indicates that the dark lamellae are structurally quite homogeneous, with collagen fibers and apatite crystals preferentially oriented parallel to the osteon axis. Bright lamellar specimens exhibit different orientation patterns with the dominant ones bidirectional at ±45° with respect to the osteon axis. Accordingly, confocal microscopy evidences the presence of longitudinal bundles in dark lamellar specimens and oblique bundles in the bright ones. Radial bundles are evidenced in both lamellar types. The alternate osteon structure is described by a rather continuous multidirectional pattern, in which dark and bright lamellae display different mechanical and possibly biological functions.

Isothermal age-hardening behaviour in a Au-1.6 wt% Ti alloy.

This study describes research with a view to developing a new age-hardenable, high-carat dental gold alloy with better biocompatibility by addition of a small quantity of titanium to gold. The relationship between isothermal age-hardening and phase transformation of the Au-1.6 wt% Ti alloy was investigated by means of hardness testing, X-ray diffraction study, scanning electron microscopic observation and energy dispersive spectroscopy. The hardening in the initial stage of ageing seemed to be attributable to the continuous precipitation of the Au4Ti ordered phase in the supersaturated alpha solid solution matrix. The overaging with softening was attributed mainly to the formation of precipitates at the grain boundaries, which grew to bright lamellae and seemed to be composed of the Au4Ti phase.

X-ray diffraction and polarizing optical microscopy investigation of the structural organization of rabbit tibia.

X-ray diffraction and polarized optical microscopy investigations were carried out on thin sections of rabbit tibia in order to study the morphological organization of the structural components of this tissue, which often is utilized to test bone response to implants. In the optical microscope, the lateral face as well as the lateral portion of the caudal face exhibit a lamellar structure with an alternation of dark and bright lamellae running parallel to the long axis of the tibia. In contrast, both in the medial face and in the medial portion of the caudal face there are numerous osteonic structures. In spite of the complexity of this morphological organization, the results of small- and high-angle X-ray diffraction analyses indicate that the structural relationship between collagen fibrils and inorganic crystals is quite similar to that observed in single osteons and allows evaluation of the orientation of the two main structural components. Both collagen fibrils and apatitic crystallites are preferentially oriented parallel to the long axis of the tibia. The degree of orientation is greater in the thickness than in the plane of the lamellae, suggesting that collagen fibrils and inorganic crystallites lie preferentially in the plane of the lamellae, where they follow an oblique course. The degree of orientation of the apatitic crystallites is higher in the lateral face than in the medial and caudal faces, in agreement with the optical microscopic images. The results provide information that must be taken into account when evaluating the structural modifications of bone due to the insertion of a prosthetic device.

Transmission electron microscopy-energy-dispersive X-ray spectroscopy analysis of the modulated structure in ZrO-6 mol% Y O alloy 2 2 3

The modulated structure formed in ZrO2-6 mol% Y2O3 alloy annealed at 1200o C for 126 h has been analysed by nanoprobe analytical electron microscopy. Transmission electron microscopy-energy-dispersive X-ray spectroscopy analysis revealed that the Y-to-Zr ratio fluctuates along a direction perpendicular to the dark and bright lamellae of the modulated structure. The fluctuation arises in a composition range between 1 and 8 mol% Y2O3 with a wavelength of about 15 nm. The dark and bright contrast is shown to be associated with the compositional fluctuation, which must be formed by spinodal decomposition.

Relationship between mechanical properties and distribution of lamellae in the human femoral shaft

The correlation between microstructure and stress distribution along a bone has always been one of the main concerns of skeletal biomechanics. By applying an original technique for isolating osteon samples and loading them under tension or compression along their axis, Ascenzi and co-workers (1, 2, 3, 4) stated that the lamellae best able to support tensile stress are those with longitudinally oriented fibre bundles (dark in cross sections observed under the polarizing microscope), while the lamellae best able to support compressive stress are those with transversely oriented fibre bundles (bright in cross sections observed under the polarizing microscope). From this result it appears clear that an analysis of the distribution and thickness of the two types of lamellae would make it possible to provide informations about the distribution of tensile vs. compressive strenght within compact bone. Such a type of analysis can be carried out using either a semiautomatic (9). or an automatic apparatus for acquisition and computation of geometric data. According to our experience the two methods give comparable results. However, an automatic apparatus is most convenient because it allows to reduce dramatically the v/orking time. In fact, the time required for analysing a cross section of a femoral diaphysis drops from two months to two hours when a semi-automatic apparatus is replaced by an automatic apparatus. So the proposal of one of us, Prof. A. Boyde, to further develop this research in his laboratory, using a Quantimet 720 image analysing computer, was accepted with enthusiasm (6, 10). The material used in this investigation was a serie. of undecalcified cross sections, 100 micra thick, each located 1 cm. from the next, and prepared using a Leitz annular blade microtome. The distribution of osteon. es and interstitial bone in terms of the proportion of their cross-sectional area taken up by bright lamellae as assessed by polarized light microscopy was done by performing a series of three operations. The first was the calculation of the surface area taken up by bright lamellae with respect to the dark background. The purpose of the second operation was to calculate the proportion of the surface actually occupied by the bone section, that is, excluding the discontinuities in the section. The aim of the third operation was to calculate what proportion of the surface of the bone section was occupied by bright lamellae through computer processing of the results of the first two operations.

Distribution of Osteonic and Interstitial Components in the Human Femoral Shaft with Reference to Structure, Calcification and Mechanical Properties

This paper analyzes the distribution of osteons and interstitial bone in the femoral compacta according to their structure, degree of calcification and mechanical properties. Three cross sections, 100 microns thick, each located 1 cm from the next, were prepared by grinding from the middle third of a human femoral shaft. Starting from the premise that, in lamellar bone, lamellae whose fiber bundles and crystallites have a longitudinal course withstand loading by tension, whereas those whose fiber bundles and crystallites have a transversal course withstand loading by compression, each osteon and fragment of interstitial bone has been given a number recording the percentage of its surface consisting of lamellae with transversally oriented fiber bundles and crystallites (bright under the polarizing microscope). The degree of calcification of the same structures was determined micro-radiographically. The distribution of both osteons and interstitial bone was assessed using a tungsten grid for reference. The total surface of each bone microstructure, and the percentage of that surface consisting of bright lamellae, were all calculated using a Zeiss Video-plan. Our results confirm the view that the distribution of both osteons and interstitial bone is mainly related to their structure--and hence to their mechanical properties. In addition, bone remodeling seems to be most active in areas capable of supporting tensile stress.

Mechanical similarities between alternate osteons and cross-ply laminates

Fully calcified osteon samples with alternating lamellae were prepared from longitudinal sections of human femoral shafts according to the technique described by Ascenzi and Bonucci. Osteons of this type reveal an alternation of dark and bright lamellae under the polarizing microscope. The samples were loaded by tension in a direction parallel with their longitudinal axis using a specially designed microwave extensimeter based on cavity and pulse techniques. All the samples were tested wet at a temperature of ca. 20°C. The stress-strain curves recorded from each osteon show a change in slope or knee at low stresses. A useful model for understanding this phenomenon is furnished by the mechanical properties of that type of fiber-reinforced plastic material called cross-ply laminate. The comparison of the data obtained from this material with those furnished by an electron microscope investigation carried out on the loaded osteons suggests that the change in slope at low stresses is due to failure of the interfibrillar cementing substance in those lamellae having fiber bundles oriented perpendicularly to the loading direction, and to yielding of the canaliculi previously filled with osteocyte processes. The physiological implications of these results are discussed.