Prolonged cyclical loading induces Haversian remodeling in mandibles of growing rabbits.

Bone adaptation to mechanical loading happens predominantly via modeling and remodeling, but the latter is poorly understood. Haversian remodeling (cortical bone replacement resulting in secondary osteons) is thought to occur in regions of low strain as part of bone maintenance or high strain in response to microdamage. However, analyses of remodeling in primates have revealed an unappreciated association with the number of daily load cycles. We tested this relationship by raising 30 male domestic rabbits (Oryctolagus cuniculus) on disparate diets from weaning to adulthood (48 weeks), facilitating a naturalistic perspective on mandibular bone adaptation. A control group consumed only rabbit pellets and an 'overuse' group ate hay in addition to pellets. To process hay, which is tougher and stiffer, rabbits increase chewing investment and duration without increasing bite force (i.e. corpus mean peak strain is similar for the two foods). Corpus remodeling in overuse rabbits was ∼1.5 times that of controls, measured as osteon population density and percentage Haversian bone. In the same subjects, there was a significant increase in overuse corpus bone formation (ratio of cortical area to cranial length), consistent with previous reports on the same dietary manipulation and bone formation in rabbits. This is the first evidence that both modeling and remodeling are simultaneously driven by the number of load cycles, independent of strain magnitude. This novel finding provides unique data on the feeding apparatus, challenges traditional thought on Haversian remodeling, and highlights the need for experimental studies of skeletal adaptation that examine mechanical factors beyond strain magnitude.

The multiscale meso-mechanics model of viscoelastic cortical bone.

Cortical bone is a complex hierarchical structure consisting of biological fiber composites with transversely isotropic constituents, whose microstructures deserve extensive study to understand the mechanism of living organisms and explore development of biomimetic materials. Based on this, we establish a three-level hierarchical structure from microscale to macroscale and propose a multiscale micromechanics model of cortical bone, which considers Haversian canal, osteonal lamellae, cement line and interstitial lamellae. In order to study the microstructural effect on the elastic behavior of hierarchical structures, the Mori-Tanaka model and locally exact homogenization theory are introduced for the homogenization of heterogeneous materials of microstructure at each level. Within sub-microscale, Haversian canal and Osteonal lamella are treated as fiber and matrix, whose homogenization is surrounded with cement line matrix in microstructure (or what we called "osteon") for the second homogenization; finally, osteon and interstitial lamella establish the meso-structure for the third homogenization, predicting the effective moduli of cortical bone. The correctness of the model in this paper is verified against the data in literature with good agreement. Finally, the dynamic viscoelastic response of cortical bones is investigated from a multiscale perspective, where the measured data are substituted into the present models to study the hydration and aging effect on bones' stiffness and viscoelasticity. It is demonstrated that the hydration is much more influential in affecting the storage and loss moduli of cortical bone than the aging effect. We also present a few numerical investigations on microstructural material and geometric parameters on the overall mechanical properties of cortical bone.

Effects of Osteocyte Shape on Fluid Flow and Fluid Shear Stress of the Loaded Bone.

This study was conducted to better understand the specific behavior of the intraosseous fluid flow. We calculated the number and distribution of bone canaliculi around the osteocytes based on the varying shapes of osteocytes. We then used these calculated parameters and other bone microstructure data to estimate the anisotropy permeability of the lacunar-canalicular network. Poroelastic finite element models of the osteon were established, and the influence of the osteocyte shape on the fluid flow properties of osteons under an axial displacement load was analyzed. Two types of boundary conditions (BC) that might occur in physiological environments were considered on the cement line of the osteon. BC1 allows free fluid passage from the outer elastic restraint boundary, and BC2 is impermeable and allows no free fluid passage from outer displacement constrained boundary. They both have the same inner boundary conditions that allow fluid to pass through. Changes in the osteocyte shape altered the maximum value of pressure gradient (PG), pore pressure (PP), fluid velocity (FV), and fluid shear stress (FSS) relative to the reference model (spherical osteocytes). The maximum PG, PP, FV, and FSS in BC2 were nearly 100% larger than those in BC1, respectively. It is found that the BC1 was closer to the real physiological environment. The fluid flow along different directions in the elongated osteocyte model was more evident than that in other models, which may have been due to the large difference in permeability along different directions. Changes in osteocyte shape significantly affect the degrees of anisotropy of fluid flow and porous media of the osteon. The model presented in this study can accurately quantify fluid flow in the lacunar-canalicular network.

Brillouin-Raman microspectroscopy for the morpho-mechanical imaging of human lamellar bone.

Bone has a sophisticated architecture characterized by a hierarchical organization, starting at the sub-micrometre level. Thus, the analysis of the mechanical and structural properties of bone at this scale is essential to understand the relationship between its physiology, physical properties and chemical composition. Here, we unveil the potential of Brillouin-Raman microspectroscopy (BRaMS), an emerging correlative optical approach that can simultaneously assess bone mechanics and chemistry with micrometric resolution. Correlative hyperspectral imaging, performed on a human diaphyseal ring, reveals a complex microarchitecture that is reflected in extremely rich and informative spectra. An innovative method for mechanical properties analysis is proposed, mapping the intermixing of soft and hard tissue areas and revealing the coexistence of regions involved in remodelling processes, nutrient transportation and structural support. The mineralized regions appear elastically inhomogeneous, resembling the pattern of the osteons' lamellae, while Raman and energy-dispersive X-ray images through scanning electron microscopy show an overall uniform distribution of the mineral content, suggesting that other structural factors are responsible for lamellar micromechanical heterogeneity. These results, besides giving an important insight into cortical bone tissue properties, highlight the potential of BRaMS to access the origin of anisotropic mechanical properties, which are almost ubiquitous in other biological tissues.

Osteon: Structure, Turnover, and Regeneration.

Bone is composed of dense and solid cortical bone and honeycomb-like trabecular bone. Although cortical bone provides the majority of mechanical strength for a bone, there are few studies focusing on cortical bone repair or regeneration. Osteons (the Haversian system) form structural and functional units of cortical bone. In recent years, emerging evidences have shown that the osteon structure (including osteocytes, lamellae, lacunocanalicular network, and Haversian canals) plays critical roles in bone mechanics and turnover. Therefore, reconstruction of the osteon structure is crucial for cortical bone regeneration. This article provides a systematic summary of recent advances in osteons, including the structure, function, turnover, and regenerative strategies. First, the hierarchical structure of osteons is illustrated and the critical functions of osteons in bone dynamics are introduced. Next, the modeling and remodeling processes of osteons at a cellular level and the turnover of osteons in response to mechanical loading and aging are emphasized. Furthermore, several bioengineering approaches that were recently developed to recapitulate the osteon structure are highlighted. Impact statement This review provides a comprehensive summary of recent advances in osteons, especially the roles in bone formation, remodeling, and regeneration. Besides introducing the hierarchical structure and critical functions of osteons, we elucidate the modeling and remodeling of osteons at a cellular level. Specifically, we highlight the bioengineering approaches that were recently developed to mimic the hierarchical structure of osteons. We expect that this review will provide informative insights and attract increasing attentions in orthopedic community, shedding light on cortical bone regeneration in the future.

Secondary osteon variants and remodeling in human bone.

Histomorphometric analysis of human cortical bone has documented the occurrence of secondary osteon variants. These include drifting osteons which form tails as they move erratically through the cortex and Type II osteons which show partial resorption and redeposition within the cement line of the osteon. Little is known about the biological significance of these variants. Prior studies suggested correlations with age, biomechanics, diet, and mineral homeostasis. No study has yet tested for osteon variant associations with static measures of bone remodeling. In this study, thin sections (n = 112) of the posterior femur representing a late English Medieval adult human osteological collection, subdivided by age, sex, and socio-economic status, were examined to determine whether remodeling indicators reconstructed from osteon parameters (area, diameter, area ratios) and densities differed between categories of presence or absence of Type II and drifting osteon variants. Of the 112 sections, 33 presented with Type II osteons, and 38 had drifting osteons. Sporadic statistically significant results were identified. Haversian canal:osteon area ratio differed (p = 0.017) with Type II osteon presence, Type II osteons were more prevalent in males than females (p = 0.048), and drifting osteons were associated with smaller osteon (p = 0.049) and Haversian canal area (p = 0.05). These results may be explained through some biological (sex) and social (status) processes such as a period of physiological recovery (e.g., following lactation, malnutrition). However, the general lack of consistent relationships between osteon variants and remodeling indicators suggests they occur as a result of natural variation.

Self-Organized Spatiotemporal Mineralization of Hydrogel: A Simulant of Osteon.

Nature creates fascinating self-organized spatiotemporal patterns through the delicate control of reaction-diffusion dynamics. As the primary unit of cortical bone, osteon has concentric lamellar architecture, which plays a crucial role in the mechanical and physiological functions of bone. However, it remains a great challenge to fabricate the osteon-like structure in a natural self-organization way. Taking advantage of the nonequilibrium reaction in hydrogels, a simple mineralization strategy to closely mimic the formation of osteon in a mild physiological condition is developed. By constructing two reverse concentration gradients of ions from periphery to interior of cylindrical hydrogel, spatiotemporal self-organization of calcium phosphate in concentric rings is generated. It is noteworthy that minerals in different layers possess diverse contents and crystalline phases, which further guide the adhesion and spread of osteoblasts on these patterns, resembling the architecture and cytological behavior of osteon. Besides, theoretical data indicates the predominate role of ion concentrations and pH values of solution, in good accordance with experimental results. Independent of precise instruments, this lifelike method is easily obtained, cost-efficient, and effectively imitates the mineral deposition in osteon from a physiochemical view. The strategy may be expanded to develop other functional material patterns via spatiotemporal self-organization.

Maximum effect of the heterogeneity of tissue mineralization on the effective cortical bone elastic properties.

The mineralization level is heterogeneous in cortical bone extracellular matrix as a consequence of remodeling. Models of the effective elastic properties at the millimeter scale have been developed based on idealizations of the vascular pore network and matrix properties. Some popular models do not take into account the heterogeneity of the matrix. However, the errors on the predicted elasticity when the difference in elastic properties between osteonal and interstitial tissues is not modeled have not been quantified. This work provides an estimation of the maximum error. We compare the effective elasticity of a representative volume element (RVE) assuming (1) different elastic properties in osteonal and interstitial tissues vs. (2) average matrix properties. In order to account for the variability of bone microstructure, we use a collection of high resolution images of the pore network to build RVEs. In each RVE we assumed a constant osteonal wall thickness and we artificially varied this thickness between 35 and 140 [Formula: see text]m to create RVEs with different amounts of osteonal tissue. The homogenization problem was solved with a fast Fourier transform (FFT)-based numerical scheme. We found that the error depends on pore volume fraction and varies on average from 1 to [Formula: see text] depending on the assumed diameter of the osteons. The results suggest that matrix heterogeneity may be disregarded in cortical bone models in most practical cases.

A Chemo-poroelastic Analysis of Mechanically Induced Fluid and Solute Transport in an Osteonal Cortical Bone.

It is well known that transport of nutrients and wastes as solute in bone fluid plays an important role in bone remodeling and damage healing. This work presents a chemo-poroelastic model for fluid and solute transport in the lacunar-canalicular network of an osteonal cortical bone under cyclic axial mechanical loading or vascular pressure. Analytical solutions are obtained for the pore fluid pressure, and fluid and solute velocities. Numerical results for fluid and calcium transport indicate that under a cyclic stress of 20 MPa, the magnitudes of the fluid and calcium velocities increase with an increase in the loading frequency for the frequency range considered (≤ 3 Hz) and peak at the inner boundary. The peak magnitude of calcium velocity reaches 18.9 µm/s for an osteon with a permeability of 1.5 × 10-19 m2 under a 3 Hz loading frequency. The magnitude of calcium velocity under a vascular pressure of 50 mmHg is found to be two orders of magnitude smaller than that under the mechanical load. These results have the potential to be important in understanding fundamental aspects of cortical bone remodeling as transport characteristics of calcium and other nutrients at the osteon scale influence bone metabolism.

Asymmetric midshaft femur remodeling in an adult male with left sided hip joint ankylosis, Metal Period Nagsabaran, Philippines.

OBJECTIVE: This study investigated microstructural changes of the right and left midshaft femur in an archaeological individual afflicted with left-sided hip joint ankylosis to assess whether increased cortical porosity was present as a result of leg disuse. MATERIALS: The individual is a middle-aged adult male excavated from the Metal Period (∼2000 BP) Nagsabaran, Luzon Island, Philippines. METHODS: Following standard examination of femur gross anatomy and differential diagnosis of the hip joint fusion, ∼1 cm thick posterior midshaft femur samples were removed for microstructural examination. Using static histomorphometry, bone multi-cellular unit activity from Haversian canal (vascular pore) density, area, and circularity was reconstructed. Spatial positioning of Haversian canals was mapped using Geographic Information Systems software. Phosphate, carbonate, and carbonate:phosphate ratios were obtained using synchrotron-sourced Fourier transform infrared microspectroscopy. RESULTS: The left femur had greater cortical pore density, with smaller and rounder vascular canals, in addition to lower matrix levels of phosphate and carbonate, when compared to the right femur. CONCLUSIONS: Our data indicate compromised bone tissue in the left femur, and conform to expected bone functional adaptation paradigms of remodeling responses to pathological and biomechanical changes. SIGNIFICANCE: The preservation of this individual's hip abnormality created a unique opportunity to evaluate intra-skeletal bone health asymmetry, which may help other researchers evaluate the presence of limb disuse in archaeological samples. LIMITATIONS: A lack of lower limb data limits our interpretations to femur remodeling only. SUGGESTIONS FOR FURTHER RESEARCH: Future research efforts should aim to examine the presence of remodeling changes in all bones of the lower limb. LAYUNIN: Gamit ang buto ng magkabilang pemur ng isang taong natagpuan sa isang archaeological site na may sakit na ankylosis sa kaliwang balakang, pinag-aralan ang iba't-ibang microstructures galing sa gitnang bahagi o midshaft ng pemur upang malaman kung may makikitang mataas na cortical porosity ang buto dahil hindi ito malimit gamitin. GAMIT: Ang pinag-aaralang buto ay galing sa isang indibidwal na tinatayang middle-age na lalaki na namuhay noong Panahon ng Metal (∼2000 BP) sa Nagsabaran, Cagayan, Republika ng Pilipinas. PAMAMARAAN: Matapos ang unang pagkilatis sa femur at ang pagkilala ng sakit sa balakang, kumuha ng ∼1 sentimetro ng buto galing sa midshaft ng pemur upang lalong mapag-aralan ang kanyang microstructure. Gamit ang static histomorphometry, napag-aralan ang mga naiwang bakas ng multi-cellular unit activity ayon sa kapal, laki at pagkakabilog ng Haversian canal (vascular pore). Gumamit din ng Geographic Information Systems (GIS) software upang mapag-aralan ang kaugnayan ng posisyon ng Haversian canal. Panghuli, gumamit din ng synchroton-sourced Fourier transform infrared (sFTIR) microspectroscopy upang makuha ang bilang ng phosphate, carbonate, at carbonate:phosphate ratio. RESULTA: Napag-alaman na ang kaliwang pemur ay mayroong higit na maraming cortical pores, maliit at mabilog na vascular canals, at mababang bilang ng phosphate, carbonate kung ihahambing sa kanang pemur. KONKLUSYON: Ayon sa aming datos, ang kaliwang pemur ay umaayon sa mga katangian ng isang butong may sakit. Sumunod din ito sa inaasahang bone functional adaptation paradigms of remodeling ng buto dahil may sakit at hindi nagamit. KAHALAGAHAN: Dahil maganda ang pagkakalibing ng buto ng balakang, nagkaroon ng pagkakataong makilatis ang kalusugan ng sinaunang-tao sa pamamagitan ng pag-aaral ng kalusugan ng buto. Dagdag pa, makakatulong din ito upang malaman kung ibang mananaliksik ang pag-aaral ng ibang butong hindi nagagamit mula sa archaeological site. LIMITASYON: Dahil walang nakuhang ibang buto mula sa binti at paa, ang pemur lang ang naimbestigahan. MUNGKAHI PARA SA MGA SUSUNOD NA PAG-AARAL: Kung magkakaroon ng pagkakataon sa susunod, dapat maimbistigahan ang lahat ng buto ng binti (lower limb).

Relation between cross-sectional bone geometry and double zonal osteon frequency and morphology.

OBJECTIVES: While double-zonal osteons (DZ) are characterized by a hyper-mineralized ring inside their lamellae, recent findings suggest that this ring is also defined by a change in the collagen fibers' orientation. Collagen and minerals are essential components to the maintenance of adequate bone strength and their alteration can modify the mechanical properties of the bone tissue. Consequently, the aim of this study is to explore the effect of past loads, as estimated from cross-sectional geometric properties, on the formation of DZ osteons compared to type I (common) osteons. MATERIALS AND METHODS: The sample consists of paired humerus and femur midshaft sections (n = 23) of Eurocanadian settlers from the historical St. Matthew cemetery, Quebec City (1771-1860). Histomorphometric variables included in this study are osteon density for DZ and type I osteons (DZD; OPD), osteon area (DZOn.Ar; On. Ar), Haversian canal area (DZH.Ar; H.Ar), and the area within the hypermineralized ring (HR. Ar). Loading history is estimated from cross-sectional properties including the following variable: cortical and total area (CA, TA), maximum and minimum second moment of area (Imax , Imin ) and polar moment of area (J). RESULTS: When the humerus and femur of the same individuals are compared, the femur has a higher OPD, DZD, and relative DZD (DZD/OPD). DZ osteons have a smaller area and Haversian canal area compared to type I osteons. The area within the hypermineralized ring in DZ is higher than the Haversian canal area of the type I osteons. Correlations between the residual scores of the regression of histomorphometric variables and cross-sectional properties of the humerus on the femur were not significant. DISCUSSION: Based on the analysis of the entire cross-section, the lack of correlation between variations in cross-sectional properties and remodeling combined with the significant differences between humeri and femura suggests that the creation of DZ or type I osteons in the bone tissue might be due to a bone specific response, possibly related to differences in bone tissue age that needs to be further investigated. Definitive conclusion regarding biomechanical loads still seem to be premature as regional variations associated with mechanical properties remain to be explored.

Network architecture strongly influences the fluid flow pattern through the lacunocanalicular network in human osteons.

A popular hypothesis explains the mechanosensitivity of bone due to osteocytes sensing the load-induced flow of interstitial fluid squeezed through the lacunocanalicular network (LCN). However, the way in which the intricate structure of the LCN influences fluid flow through the network is largely unexplored. We therefore aimed to quantify fluid flow through real LCNs from human osteons using a combination of experimental and computational techniques. Bone samples were stained with rhodamine to image the LCN with 3D confocal microscopy. Image analysis was then performed to convert image stacks into mathematical network structures, in order to estimate the intrinsic permeability of the osteons as well as the load-induced fluid flow using hydraulic circuit theory. Fluid flow was studied in both ordinary osteons with a rather homogeneous LCN as well as a frequent subtype of osteons-so-called osteon-in-osteons-which are characterized by a ring-like zone of low network connectivity between the inner and the outer parts of these osteons. We analyzed 8 ordinary osteons and 9 osteon-in-osteons from the femur midshaft of a 57-year-old woman without any known disease. While the intrinsic permeability was 2.7 times smaller in osteon-in-osteons compared to ordinary osteons, the load-induced fluid velocity was 2.3 times higher. This increased fluid velocity in osteon-in-osteons can be explained by the longer path length, needed to cross the osteon from the cement line to the Haversian canal, including more fluid-filled lacunae and canaliculi. This explanation was corroborated by the observation that a purely structural parameter-the mean path length to the Haversian canal-is an excellent predictor for the average fluid flow velocity. We conclude that osteon-in-osteons may be particularly significant contributors to the mechanosensitivity of cortical bone, due to the higher fluid flow in this type of osteons.

Age-related properties at the microscale affect crack propagation in cortical bone.

The increased risk for fracture with age is associated not only with reduced bone mass but also with impaired bone quality. At the microscale, bone quality is related to porosity, microstructural organization, accumulated microdamage and intrinsic material properties. However, the link between these characteristics and fracture behavior is still missing. Bone tissue has a complex structure and as age-related compositional and structural changes occur at all hierarchical length scales it is difficult to experimentally identify and discriminate the effect of each mechanism. The aim of this study was therefore to use computational models to analyze how microscale characteristics in terms of porosity, intrinsic toughness properties and microstructural organization affect the mechanical behavior of cortical bone. Tensile tests were simulated using realistic microstructural geometries based on microscopy images of human cortical bone. Crack propagation was modelled using the extended finite element method where cement lines surrounding osteons were modelled with an interface damage law to capture crack deflections along osteon boundaries. Both increased porosity and impaired material integrity resulted in straighter crack paths with cracks penetrating osteons, similar to what is seen experimentally for old cortical bone. However, only the latter predicted a more brittle failure behavior. Furthermore, the local porosity influenced the crack path more than the macroscopic porosity. In conclusion, age-related changes in cortical bone affect the crack path and the mechanical response. However, increased porosity alone was not driving damage in old bone, but instead impaired tissue integrity was required to capture brittle failure in aging bone.

Haversian remodeling corresponds to load frequency but not strain magnitude in the macaque (Macaca fascicularis) skeleton.

One way bone adapts to its mechanical environment is by Haversian remodeling, a repair process in which existing bone is resorbed and replaced by new bone. Haversian remodeling forms interconnected, cylindrical structures called secondary osteons. The amount of remodeling that occurs is related to the nature of mechanical loading and accrual of microdamage, but it is uncertain whether habitual loads of high magnitude versus high frequency result in more remodeling. The answer to this question is important if remodeling is to be a tool for inferring loading environments, and thus behavior, in past populations. Here, secondary osteon population density (OPD), osteon cross-sectional area (On.Ar), and percent Haversian bone (%HAV) were compared among mid-diaphysis femora, tibia, fibulae, and mid-level ribs of five adult crab-eating macaques (Macaca fascicularis). Ribs experience relatively low strains but have a high daily loading frequency (~33 times per minute). Limb bones are loaded for fewer cycles per day, but the femur and tibia have high load magnitudes due to gravitational forces. Strain magnitudes in the fibula are a fraction of those in the femur and tibia. Analyses of variance demonstrated significant differences in OPD (P = 0.010) and On.Ar (P < 0.001) among the bones. Pairwise t-tests revealed greater OPD but lower On.Ar in the rib than all other bones. The high rib OPD suggests that Haversian remodeling is more responsive to load frequency than strain magnitude. The fact that osteons are smaller in ribs than any other bone may be an effect of remodeling in comparatively narrow cortices.

Bioprinted osteon-like scaffolds enhance in vivo neovascularization.

Bone tissue engineers are facing a daunting challenge when attempting to fabricate bigger constructs intended for use in the treatment of large bone defects, which is the vascularization of the graft. Cell-based approaches and, in particular, the use of in vitro coculture of human umbilical vein endothelial cells (HUVECs) and human mesenchymal stem cells (hMSCs) has been one of the most explored options. We present in this paper an alternative method to mimic the spatial pattern of HUVECs and hMSCs found in native osteons based on the use of extrusion-based 3D bioprinting (3DP). We developed a 3DP biphasic osteon-like scaffold, containing two separate osteogenic and vasculogenic cell populations encapsulated in a fibrin bioink in order to improve neovascularization. To this end, we optimized the fibrin bioink to improve the resolution of printed strands and ensure a reproducible printing process; the influence of printing parameters on extruded strand diameter and cell survival was also investigated. The mechanical strength of the construct was improved by co-printing the fibrin bioink along a supporting PCL carrier scaffold. Compressive mechanical testing showed improved mechanical properties with an average compressive modulus of 131 ± 23 MPa, which falls in the range of cortical bone. HUVEC and hMSC laden fibrin hydrogels were printed in osteon-like patterns and cultured in vitro. A significant increase in gene expression of angiogenic markers was observed for the biomimetic scaffolds. Finally, biphasic scaffolds were implanted subcutaneously in rats. Histological analysis of explanted scaffolds showed a significant increase in the number of blood vessels per area in the 3D printed osteon-like scaffolds. The utilization of these scaffolds in constructing biomimetic osteons for bone regeneration demonstrated a promising capacity to improve neovascularization of the construct. These results indicates that proper cell orientation and scaffold design could play a critical role in neovascularization.

Understanding age-induced cortical porosity in women: Is a negative BMU balance in quiescent osteons a major contributor?

Cortical bone is remodeled by intracortical basic multicellular units (BMUs), whose end result can be observed as quiescent osteons in histological sections. These osteons offer a unique opportunity to investigate the BMU balance between the magnitude of bone resorption and subsequent bone formation at the BMU level. Our main objective was to investigate whether the latter parameters change between defined categories of osteons and with age, and to which extend these changes contribute to age-induced cortical porosity. Cortices of iliac bone specimens from 35 women (aged 16-78 years) with a higher porosity with age were investigated. A total of 3084 quiescent osteons reflecting 75% of the intracortical pores were histological examined. The osteons diameter, pore diameter, wall thickness, prevalence and contribution to the porosity were highly variable, but unchanged with age. Next, the osteons were categorized according to whether they reflected the remodeling of existing canals (type 2Q osteons) or the generation of new canals (type 1Q osteons). Type 2Q osteons versus type 1Q osteons: (i) had more frequently a pore diameter > 75 µm (7.4 vs. 1.3%; p < 0.001); (ii) had a larger mean pore diameter (40 ±â€¯10 vs. 25 ±â€¯4 µm; p < 0.001), osteon diameter (120 ±â€¯21 vs. 94 ±â€¯21 µm; p < 0.001) and wall thickness (40 ±â€¯10 vs. 35 ±â€¯9; p < 0.05); (iii) had a larger contribution to the cortical porosity (29 ±â€¯18 vs. 8 ±â€¯8%; p < 0.001); (iv) were more prevalent (44 ±â€¯10 vs. 31 ±â€¯11%; p < 0.001); and (v) were more prevalent with age. Collectively, this study demonstrates that quiescent osteons with age more frequently result from remodeling of existing canals, which in some cases had a more negative BMU balance. Still, the osteons showed no overall age-related change in their pore diameter i.e. BMU balance. In contrast to conventional wisdom, these data show that non-quiescent pores, not pores of quiescent osteons, were the main contributor to a higher cortical porosity.

Diversity in intracortical remodeling in the human femoral bone: A novel view point with the morphological analysis of secondary osteons.

INTRODUCTION: In humans, intracortical bone remodeling is performed by a basic multicellular unit (BMU) composed of osteoclasts and osteoblasts penetrating through cortical bones. As a result, secondary osteons and their boundaries, cement lines, can be observed on the transverse section. There have been few reports mention whether there is diversity within a single individual and on the relevance to bone remodeling. The purpose of this study is to investigate the morphological diversity of secondary osteons in human femoral bone and to examine the relationship with bone remodeling. MATERIAL AND METHODS: First of all, we developed an original method to get the cross-sectional images of the cortical bones around the whole circumference for the purpose of evaluating the morphology of the secondary osteon exhaustively. Then, a total of ten cross-sectional slices from one right human femoral bone of male were prepared and stained with this method. The osteon population density and complexity of cement lines in osteons were evaluated in detail. RESULTS: Within this femoral bone, the osteon population density was significantly higher in the periosteal side and in the posterior area. Conversely, the cement line density and the osteon complexity were higher in the endosteal side; the proportion of complexed osteon significantly increased from the periosteal side toward the endosteal side. DISCUSSION: The results suggested that there were diversities in osteon population densities and osteon morphological pattern within one human femoral bone. It seemed that the BMUs ran to avoid the existing regions of osteon in the periosteal sides and to overlap the existing osteon in the endosteal sides. This seemed to be one of the novel viewpoints in the morphological analysis of secondary osteons. It might be better for the orthopedic surgeons to be aware that the osteon distribution in the cortical bone is not uniform.

A multi-layered poroelastic slab model under cyclic loading for a single osteon.

BACKGROUND: An osteon consists of a multi-layered bone matrix and interstitial fluid flow in the lacunar-canalicular system. Loading-induced interstitial fluid flow in the lacunar-canalicular system is critical for osteocyte mechanotransduction and bone remodelling. METHODS: To investigate the effects of the lamellar structure and heterogeneous material properties of the osteon on the distributions of interstitial fluid flow and seepage velocity, an osteon is idealized as a hollow two-dimensional poroelastic multi-layered slab model subjected to cyclic loading. Based on poroelastic theory, the analytical solutions of interstitial fluid pressure and seepage velocity in lacunar-canalicular pores were obtained. RESULTS: The results show that strain magnitude has a greater influence on interstitial fluid pressure than loading frequency. Interestingly, the heterogeneous distribution of permeability produces remarkable variations in interstitial fluid pressure and seepage velocity in the cross-section of cortical bone. In addition, interstitial fluid flow stimuli to osteocytes are mostly controlled by the value of permeability at the surface of the osteon rather than at the inner wall of the osteon. CONCLUSION: Interstitial fluid flow induced by cycling loading stimuli to an osteocyte housed in a lacunar-canalicular pore is not only correlated with strain amplitude and loading frequency, but also closely correlated with the spatial gradient distribution of permeability. This model can help us better understand the fluid flow stimuli to osteocytes during bone remodelling.

Intracortical stiffness of mid-diaphysis femur bovine bone: lacunar-canalicular based homogenization numerical solutions and microhardness measurements.

Microscale lacunar-canalicular (L-C) porosity is a major contributor to intracortical bone stiffness variability. In this work, such variability is investigated experimentally using micro hardness indentation tests and numerically using a homogenization scheme. Cross sectional rings of cortical bones are cut from the middle tubular part of bovine femur long bone at mid-diaphysis. A series of light microscopy images are taken along a line emanating from the cross-section center starting from the ring's interior (endosteum) ring surface toward the ring's exterior (periosteum) ring surface. For each image in the line, computer vision analysis of porosity is conducted employing an image segmentation methodology based on pulse coupled neural networks (PCNN) recently developed by the authors. Determined are size and shape of each of the lacunar-canalicular (L-C) cortical micro constituents: lacunae, canaliculi, and Haversian canals. Consequently, it was possible to segment and quantify the geometrical attributes of all individual segmented pores leading to accurate determination of derived geometrical measures such as L-C cortical pores' total porosity (pore volume fraction), (elliptical) aspect ratio, orientation, location, and number of pores in secondary and primary osteons. Porosity was found to be unevenly (but linearly) distributed along the interior and exterior regions of the intracortical bone. The segmented L-C porosity data is passed to a numerical microscale-based homogenization scheme, also recently developed by the authors, that analyses a composite made up of lamella matrix punctuated by multi-inclusions and returns corresponding values for longitudinal and transverse Young's modulus (matrix stiffness) for these micro-sized spatial locations. Hence, intracortical stiffness variability is numerically quantified using a combination of computer vision program and numerical homogenization code. These numerically found stiffness values of the homogenization solution are corroborated experimentally using microhardness indentation measurements taken at the same points that the digital images were taken along a radial distance emanating from the interior (endosteum) surface toward the bone's exterior (periosteum) surface. Good agreement was found between numerically calculated and indentation measured stiffness of Intracortical lamellae. Both indentation measurements and numerical solutions of matrix stiffness showed increasing linear trend of compressive longitudinal modulus (E11) values vs. radial position for both interior and exterior regions. In the interior (exterior) region of cortical bone, stiffness modulus values were found to range from 18.5 to 23.4 GPa (23 to 26.0 GPa) with the aggregate stiffness of the cortical lamella in the exterior region being 12% stiffer than that in the interior region. In order to further validate these findings, experimental and FEM simulation of a mid-diaphysis bone ring under compression is employed. The FEM numerical deflections employed nine concentric regions across the thickness with graded stiffness values based on the digital segmentation and homogenization scheme. Bone ring deflections are found to agree well with measured deformations of the compression bone ring.

Coalignment of osteocyte canaliculi and collagen fibers in human osteonal bone.

During bone formation osteocytes get connected with each other via a dense network of canaliculi within the mineralized bone matrix. Important functions attributed to the osteocyte network include the control of bone remodeling and a contribution to mineral homeostasis. To detect structural clues of the formation and functionality of the network, this study analyzes the structure and orientation of the osteocyte lacuno-canalicular network (OLCN), specifically in relation to the concentric bone lamellae within human osteons. The network structure within 49 osteons from four samples of cortical bone from the femoral midshaft of middle-aged healthy women was determined by a combination of rhodamine staining and confocal laser scanning microscopy followed by computational image analysis. A quantitative evaluation showed that 64±1% of the canalicular length has an angle smaller than 30° to the direction towards the osteon center, while the lateral network - defined by an orientation angle larger than 60° - comprises 16±1%. With the same spatial periodicity as the bone lamellae, both radial and lateral network show variations in the network density and order. However, only the preferred orientation of the lateral network twists when crossing a lamella. This twist agrees with the preferred orientation of the fibrous collagen matrix. The chirality of the twist was found to be individual-specific. The coalignment between network and matrix extends to the orientation of the elongated osteocyte lacunae. The intimate link between OLCN and collagen matrix implies an interplay between osteocyte processes and the arrangement of the surrounding collagen fibers during osteoid formation.