Single Osteons Research Articles

A microstructure-based study on compact human bones: hierarchical length scale parameter

This paper presents a microstructure-based study on compact human bone using the modified couple stress theory and investigates some of its mechanical behaviors at the micro-scale. First of all, length scale parameters in torsion and bending are assessed for bone micro-samples on the basis of experimental data proposed by Yang and Lakes. Then, the effect of micro-rotations on the torsion and bending of single osteons is studied and compared with experimental results obtained by Ascenzi et al., and the length scale parameter of osteons is consequently obtained. According to the present study on osteons and using an approximate formulation of multi-layered tubes under torsion, a material length scale parameter of lamellae is acquired to capture the size effect. These achievements lead to introducing a hierarchical structure for the material length scale parameter of the modified couple stress theory. In addition, the buckling, static nonlinear response, torsional vibration, and flexural dispersion analyses of osteons are presented.

OS7-2-1 Estimation of Viscoelastic Properties of Rat Cortical Bone Using Dynamic Nanoindentation

The objective of this study was to use the dynamic nanoindentation method to determine viscoelastic properties such as storage modulus and loss tangent of single osteons and interstitial lamellae of rat femoral cortical bone tissue at the microstructural level in a longitudinal direction. The effects of mineral, organic and water contents of bone specimens on viscoelastic properties at the macroscopic level were also investigated. The storage moduli of osteons were significantly lower than those of interstitial lamellae, however, the loss tangents of osteons were higher than those of interstitial lamellae of bone tissues. A correlation was found between the storage modulus and mineral content of rat cortical specimens. Likewise, a linear correlation was also demonstrated between the loss tangent and the organic and water contents of specimens.

Mechanics of bone tissue revisited from nano to macro

In a biomechanical perspective, the clinically relevant concept of bone quality is essentially determined by the material properties of bone tissue. What are these material properties and how are they affected by growth, remodeling, aging, disease, treatment and healing? Despite significant advances of the field in the past decades, these questions have not been answered completely. Bone tissue is a hierarchical composite including a mineral phase, a collageneous phase and water. Beyond these heteroclite molecular constituents, bone tissue exhibits three major levels of organisation that determine its macroscopic mechanical behaviour. The first level of organisation, the mineralized collagen fibril represents the most challenging level from the experimental point of view. The arrangement and bonds of the few nanometer thick hydroxyapatite platelets with the cross-linked collagen fibrils appear to be the key to understand deformation and load transfer in this elementary unit. Damage necessarily initiates at this level and osteointegration depends on the resulting surface chemistry. The lamella represents the second level of organisation and was mainly investigated by scanning nanoindentation. The rotated plywood configuration of the lamellae controls the orientation of the mineralized collagen fibrils and inhibits microcrack propagation. However, mechanical-morphological relationships are still missing at this level. The third level is the bone structural unit (BSU) resulting from the bone remodeling process. These BSU exhibit heterogeneous mineralization, damage and microcrack densities. While single osteons have been previously tested in tension, compression and torsion, most studies interested in the influence of diet, pharmacological treatment or gene mutation on BSU mechanics were performed using nanoindentation. The compact and trabecular microstructures constitute the macrosopic level. While poroviscoelasticity, damage and fracture of bone tissue have been the object of an impressive number of studies, it remains unclear how these properties are quantitatively affected by the lower levels of organisation. In a clinical perspective, material models of bone tissue are developed to compute the mechanical behaviour of whole organs that will hopefully contribute to improve prevention and treatment of osteoporosis and other bone diseases.

Osteocytic Expression of Constitutive NO Synthase Isoforms in the Femoral Neck Cortex: A Case-Control Study of Intracapsular Hip Fracture

NO is an osteocytic signaling molecule that can inhibit osteoclasts. The NO synthases eNOS and nNOS were expressed by >50% of osteonal osteocytes in controls. Hip fracture cases showed +NOS osteocytes only in deep osteonal bone, and 25-35% reduced expression overall. These data are consistent with increased osteonal vulnerability to deep osteoclastic attack. Osteocytes may regulate the response to mechanical stimuli in bone through the production of local signaling molecules such as NO derived from the NO synthase eNOS. Because NO is inhibitory to osteoclastic resorption, it has been suggested that osteocytes expressing eNOS act as sentinels, confining resorption within single osteons. Recently, nNOS has been shown to be present in osteocytes of adult human bone. Cross-sections of the femoral neck (eight female cases of intracapsular hip fracture and seven postmortem controls; age, 68-91 years) were analyzed by immunohistochemistry. The percentages of osteocytes expressing each of these two isoforms were calculated, and their distances to the nearest canal surface were measured. The percentage of +nNOS osteocytes was lower in the fracture cases than in the controls (cases: 43.12 +/- 1.49, controls: 56.68 +/- 1.45; p < 0.0001). Compared with nNOS, eNOS expression was further reduced (p = 0.009) in the cases but was not different in the controls (cases: 36.41 +/- 1.53, controls: 56.47 +/- 2.41; p < 0.0001). The minimum distance of +eNOS or +nNOS osteocytes to a canal surface was higher in the cases compared with controls (eNOS: controls; 44.4 +/- 2.2 microm, cases: 61.7 +/- 2.0 microm; p < 0.0001; nNOS: controls: 52.4 +/- 1.7 microm, cases: 60.2 +/- 2.1 microm; p = 0.0039). +eNOS osteocytes were closer to the canal surfaces than +nNOS osteocytes in the controls by 8.00 +/- 4.0 microm (p = 0.0012). The proportions of osteocytes expressing nNOS and eNOS were both reduced in the fracture cases, suggesting that the capacity to generate NO might be reduced. Furthermore, the reduction in NOS expression occurs in those osteocytes closest to the canal surface, suggesting that the ability of NO to minimize resorption depth might be impaired. Further studies are needed on the regulation of the expression and activity of these distinct NOS isoforms.

Mathematical modeling of human secondary osteons.

This investigation explores the structural dimensions and patterns within single secondary osteons, with consideration of their biological variation. New data from images obtained previously of osteons observed through linearly polarized light, electron microscopy, and micro-x-ray, combined with recent findings on lamellae by circularly polarized light, confocal microscopy, synchrotron x-ray diffraction, and micro-x-ray, provide the basis for novel computerized models of single osteons and single lamellae. The novelty of such models is the concurrent representation of (1) collagen-hydroxyapatite orientation, (2) relative hydroxyapatite percentage, (3) distributions of osteocytes' lacunae and canaliculae, and (4) biological variations in dimensions of the relevant structures. The mathematical software Maple realizes the computerized models. While the parts of the models are constructed on a personal computer, the voluminous data associated with the representation of lacunar and canalicular distributions require a supercomputer for assembly of the models and final analysis. The programming used to define the models affords the option to randomize the dimensional specifications of osteons, lamellae, lacunae, and canaliculae within the experimentally observed numeric ranges and distributions. Through this option, the program can operate so that each run of the file produces a unique random model within the observed biological variations. The program can also be run to implement specific dimensional requirements. The modeling has applications in the microstructural study of fracture propagation and remodeling, as well as in the simulation of mechanical testing. The approach taken here is of wide application and could be of value in other areas of microscopy such as scanning electron microscopy, microcomputerized tomography scan, and magnetic resonance imaging on cancellous bone structures.

Patterns of osteocytic endothelial nitric oxide synthase expression in the femoral neck cortex: differences between cases of intracapsular hip fracture and controls

Evidence indicates that extensive amalgamation of adjacent resorbing osteons is responsible for destroying the microstructural integrity of the femoral neck’s inferior cortex in osteoporotic hip fracture. Such osteonal amalgamation is likely to involve a failure to limit excessive resorption, but its mechanistic basis remains enigmatic. Nitric oxide (NO) inhibits osteoclastic bone destruction, and in normal bone cells its generation by endothelial nitric oxide synthase (eNOS, the predominant bone isoform) is enhanced by mechanical stimuli and estrogen, which both protect against fracture. To determine whether eNOS expression in osteocytes reflects their proposed role in regulating remodeling, we have examined patterns of osteocyte eNOS immunolabeling in the femoral neck cortex of seven cases of hip fracture and seven controls (females aged 68–96 years). The density of eNOS + cells (mm −2) was 53% lower in the inferior cortex of the fracture cases ( p < 0.0004), but was similar in the superior cortex. eNOS + osteocytes were, on average, 22% further from their nearest blood supply, than osteocytes in general ( p < 0.0001) and the nearest eNOS + osteocyte was 57% further from its nearest canal surface ( p < 0.0001). This differential distribution of eNOS + osteocytes was significantly more pronounced in the cortices of fracture cases ( p < 0.0001). We conclude that the normal regional and osteonal pattern of eNOS expression by osteocytes is disrupted in hip fracture, particularly at sites that are loaded most by physical activity. These results suggest that eNOS + osteocytes may normally act as sentinels confining resorption within single osteons. A reduction in their number, coupled to an increase in their remoteness from canal surfaces, may thus permit the irreversible merging of resorbing osteons, and thus contribute to the marked increase in the fragility of osteoporotic bone.

The mandibular canal of the edentulous jaw.

The morphology of the mandibular canal after loss of teeth has received little detailed attention. Improved documentation of this topic would allow better interpretation of dental radiographs and would enable those engaged in tooth implantation to better understand the nature of the tissue into which the prostheses are placed. In this study on mandibles from seven dissecting room cadavers panoramic radiographs usually showed the mandibular canal clearly, an incisive canal less so. The wall of the mandibular canal was similar in dentate and edentulous mandibles, and was highly perforated, as suggested by Cryer (Anderson et al., 1991). In edentulous specimens, it was composed mainly of cancellous bone with only occasional single osteons. The inferior alveolar nerve near the mandibular foramen was a large trunk, consisting of three to four nerve bundles with connective tissue sheaths. It became more loosely arranged toward the mental foramen. Medial to the mental foramen, the nerves were frequently in the form of small bundles in the marrow. Any incisive canal was ill-defined and neurovascular bundles, when present, ran through a labyrinth of intertrabecular spaces.

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.

X-Ray Diffraction on Cyclically Loaded Osteons

The results of a study on the fine structural distortion due to the two previously observed types of degradation in cyclically loaded single osteons (i.e., stiffness degradation and pinching effect) are presented. Fully calcified longitudinal and alternate osteons were isolated from 350-microns-thick longitudinal sections of human femoral cortical bone. The samples were prepared from 500-microns-long central cylindrical portions of an osteon, whose two ends were penetrating into rectangular lugs for fixation to an electromechanical device that cyclically loaded the samples. This device was connected to a microwave micrometer and a recorder. The structural distortions induced by cyclic loading were investigated by high- and low-angle X-ray diffraction on conventional and synchrotron radiation sources. Cyclic loading results in a reduction in the degree of orientation of apatite crystallites, especially in longitudinal osteons, in which the most abundant longitudinal lamellae are not protected against buckling by transverse lamellae as they are in alternate osteons. In contrast, the degree of orientation of collagen fibrils does not seem to be affected by cycling loading in the two osteon types, possibly because the disorientation of collagen fibrils is, within limits, a reversible process. Finally, the contrast between the disorientation of inorganic crystallites and the apparently unaltered distribution of collagen fibrils suggests that the degradation of cyclically loaded osteons may be due to a separation of the crystallites from the fibrils.

Elastic properties of human cortical and trabecular lamellar bone measured by nanoindentation

An experimental investigation was undertaken to measure the intrinsic elastic properties of several of the microstructural components of human vertebral trabecular bone and tibial cortical bone by the nanoindentation method. Specimens from two thoracic vertebrae (T-12) and two tibiae were obtained from frozen, unembalmed human male cadavers aged 57 and 61 years. After drying and mounting in epoxy resin, nanoindentation tests were conducted to measure Young's modulus and the hardness of individual trabeculae in the vertebrae and single osteons, and interstitial lamellae in the tibiae. Measurements on the vertebral trabeculae were made in the transverse direction, and the average Young's modulus was found to be 13.5 ± 2.0 GPa. The tibial specimens were tested in the longitudinal direction, yielding moduli of 22.5 ± 1.3 GPa for the osteons and 25.8 ± 0.7 GPa for the interstitial lamellae. Analysis of variance showed that the differences in the measured moduli are statistically significant. Hardness differences among the various microstructural components were also observed.

Scanning Acoustic Microscope Studies of the Elastic Properties of Osteons and Osteon Lamellae

Scanning acoustic microscopy (SAM) provides the means for studying the elastic properties of a material at a comparable level of resolution to that obtained by optical microscopy for structural studies. SAM is nondestructive and permits observation of properties in the interior of materials which are optically opaque. Two modes of ultrasonic signals have been used in a Model UH3 Scanning Acoustic Microscope (Olympus Co., Tokyo, Japan) as part of a continuing study of the microstructural properties of bone. The pulse mode, using a single narrow pulse in the range of 30 MHz to 100 MHz, has been used to survey the surface and interior of specimens of human and canine femoral compact cortical bone at resolutions down to approximately 30 microns. To obtain more detailed information at significantly higher resolution, the burst mode, comprised of tens of sinusoids, has been used at frequencies from 200 MHz to 600 MHz. This has provided details of both human and canine single osteons (or haversion systems) and ostenoic lamellae at resolutions down to approximately 1.7 microns, well within the thickness of a lamella as viewed in a specimen cut transverse to the femoral axis.

The bending properties of single osteons

The bending properties of two fully calcified osteon types (longitudinal and alternate) have been investigated in 62 cylindrical samples by applying the technique of three-point bending loading. The bending of each sample was recorded using a microwave micrometer based on the cavity and pulse technique. It has been shown that alternate osteons are better able to withstand bending stress than longitudinal ones. This result offers a definitive explanation for the high concentration of transverse lamellae in pathologically bowed bone shaft.

Calcified turkey leg tendon as structural model for bone mineralization

Samples of turkey tendon at different degree of calcification have been investigated by high and small angle X-ray diffraction techniques using conventional and synchrotron radiation sources. The results indicate that the small angle diffraction patterns of samples at a degree of calcification ranging from 20 to 60% wt are due to the apatite phase which fits in the main band of collagen fibrils and exhibits the same axial D-periodicity, 67.0 nm, as native collagen fibrils in either wet or air-dried conditions. Analysis of these patterns reveals that the mineral phase is deposited along the collagen fibrils according to a step function. The length of the step is 0.46 D independent of the degree of calcification, while the height of the step increases with increasing calcification. The shift of the high angle equatorial diffraction spacing from 1.42 to 1.24 nm when the inorganic phase content passes from 40 to 60% wt suggests that needle-shaped crystals which compress the molecules are preferentially deposited during the advanced stages of calcification, in agreement with previuos electron microscopy observations. The structural relationship between inorganic deposits and collagen matrix is similar to that found for single osteons. This similarity increases as calcification proceeds.

Mechanical hysteresis loops from single osteons: Technical devices and preliminary results

The purpose of this paper is to describe an original apparatus for recording hysteresis loops from single osteons and a special microgrinding lathe for preparing osteonic samples for testing. The results obtained by testing isolated osteons, and composite bone samples ground to the same size as an osteon sample justify one to draw the following main conclusions: (a) at an equal degree of calcification, longitudinal osteons show larger hysteresis loops under compression and alternate osteons show larger hysteresis loops under tension; (b) there seems to be little chance of acquiring detailed information on the mechanical effects of osteon calcification recording hysteresis loops; (c) collagen orientation in lamellae is the main factor determining the kind of hysteresis loops displayed by a composite bone sample.

Strain and frequency dependence of shear storage modulus for human single osteons and cortical bone microsamples—Size and hydration effects

Using a specially designed and laboratory built microtorsional device, the shear storage modulus of single osteons and microsamples containing up to a dozen osteons was investigated for both strain and frequency dependence. The data indicate that an increase in number of osteons in the microsample reduces the storage modulus, and results in smaller strains at which deviation from linearity and plasticity effects are first noticed. Size effects were also noticed, which suggest that an increase in number of osteons, increases the effectiveness of water (hydration of dry samples) in reducing the storage modulus and in increasing the internal energy loss in the sample. Some of the effects were noticed to approach saturation in samples with more than a half dozen osteons, while others appeared to persist (at least up to a dozen osteons). Also noticed, were reductions in shear storage modulus of 10–35% over an approximate frequency reduction of 10 3–1 Hz for microsamples containing one or more osteons.

Strain dependence of dynamic Young's modulus for human single osteons

The strain dependence of Young's modulus for human single osteons was investigated at frequencies near 10 3 Hz by means of a small acoustic speaker. On average, deviations from linear dependence occurred at 2% strain and plasticity followed at 4% strain. The modulus itself averaged at 12–14 GN/m 2 and is comparable to values obtained both by Black (1972) and by Smith and Keiper (1965) for samples containing many osteons at frequencies of 350 and 500–3500 Hz respectively.

Theoretical evidence for the generation of high pressure in bone cells

A simple model of the mechanical behavior of the bone cell-bone matrix composite has been used to compute the hydrostatic pressure which may be developed within osteocytes when bone is placed under stress. Using current estimates of the elastic properties of long bones and single osteons it is shown that predicted pressures are within the range known to affect cellular function.

Isolation of single osteons and osteon lamellae.

Single human osteons were isolated by propagating fractures along their natural boundaries. Furthermore, decalcified osteons were mechanically manipulated to expose their interlamellar interfaces, making possible the study of collagen fibers by means of scanning electron microscopy (SEM). Isolation of single osteons of a wide range of lengths, as described in this paper, makes possible for the first time the study of their mechanical properties in all possible modes. SEM studies of exposed lamellar interfaces reveal that collagen fiber orientations are more complex than previously suggested and suggest further studies in an effort to solve past controversies on collagen fiber orientations in human bone.

Scanning and transmission electron microscopy of calcified tissues.

Some recent innovations have been utilized to extend studies of the structure of calcified tissues on the ultra- and microstructural levels of organization. The introduction of hydrazine as a replacement for the conventionally used ethylene diamine to deproteinize calcified tissue samples has resulted in the acquisition of previously unobtainable information about the mineral phase as it exists in situ. This enables inference of mineral-organic interrelationships when coupled with the EDTA demineralization technique used to study collagen fiber frameworks. In addition, a method for obtaining whole single osteons and separating them into lamellar constituents permits observations of the collagen-apatite orientation within successive lamellae of compact Haversian bone. The use of the structural information derived to develop a fiber-reinforced model of compact bone as a composite material is outlined and discussed.

The shearing properties of single osteons.

AbstractThe shearing strength of single human osteons has been investigated using a microtesting machine equipped with a microwave micrometer. The results Were related to the degree of calcification and the orientation of collagen fiber bundles in successive lamellae of the osteons. The following conclusions were made: (1) Osteons having a marked longitudinal spiral course of fiber bundles in successive lamellae are least able to support shearing stress. This suggests that in the other types of osteon the compactness of bone is strengthened by the lamellae having marked transversal spiral course of the fiber bundles. (2)Ultimate shearing strength and modulus of elasticity of osteons increase as calcification proceeds. (3) The shearing strength of single osteons is markedly lower than the tensile and compressive strength for samples of the same type. (4) In osteons loaded along their axis the range of elastic deformation is barely more than 1% of the length of the sample. (5) With the technical procedure used in this investigation the shearing of osteons appears to be preferentially related to the lamellar structure. In osteons loaded excentrically the portions which have slipped out have a rather irregular shape and in many cases one or two fractures occur. (6) The resistance to shearing of the cementing substance at the boundaries of the osteon may be greater than the resistance of the osteon itself.