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.

Osteoblasts infill irregular pores under curvature and porosity controls: a hypothesis-testing analysis of cell behaviours.

The geometric control of bone tissue growth plays a significant role in bone remodelling, age-related bone loss, and tissue engineering. However, how exactly geometry influences the behaviour of bone-forming cells remains elusive. Geometry modulates cell populations collectively through the evolving space available to the cells, but it may also modulate the individual behaviours of cells. To factor out the collective influence of geometry and gain access to the geometric regulation of individual cell behaviours, we develop a mathematical model of the infilling of cortical bone pores and use it with available experimental data on cortical infilling rates. Testing different possible modes of geometric controls of individual cell behaviours consistent with the experimental data, we find that efficient smoothing of irregular pores only occurs when cell secretory rate is controlled by porosity rather than curvature. This porosity control suggests the convergence of a large scale of intercellular signalling to single bone-forming cells, consistent with that provided by the osteocyte network in response to mechanical stimulus. After validating the mathematical model with the histological record of a real cortical pore infilling, we explore the infilling of a population of randomly generated initial pore shapes. We find that amongst all the geometric regulations considered, the collective influence of curvature on cell crowding is a dominant factor for how fast cortical bone pores infill, and we suggest that the irregularity of cement lines thereby explains some of the variability in double labelling data as well as the overall speed of osteon infilling.

Bone tissue aging affects mineralization of cement lines.

Cement lines are known as thin peripheral boundaries of the osteons. With a thickness below 5 µm their composition of inorganic and organic compounds has been a matter of debate. Here, we hypothesized that cement lines become hypermineralized and their degree of mineralization is not constant but related to the tissue age of the osteon. Therefore, we analyzed the calcium content of osteons and their corresponding cement lines in a range of different tissue ages reflected by osteonal mineralization levels in femoral cortical bone of both postmenopausal women with osteoporosis and bisphosphonate-treated cases. Quantitative backscattered electron imaging (qBEI) showed that cement lines are hypermineralized entities with consistently higher calcium content than their corresponding osteons (mean calcium content: 29.46 ±â€¯0.80 vs. 26.62 ±â€¯1.11 wt%; p < 0.001). Micro-Raman spectroscopy complemented the qBEI data by showing a significantly higher phosphate/amide I ratio in the cement lines compared to the osteonal bone (8.78 ±â€¯0.66 vs. 6.33 ±â€¯0.58, p < 0.001), which was both due to an increased phosphate peak and a reduced amide I peak in cement lines. A clear positive correlation of cement line mineralization and the mineralization of the osteon was observed (r = 0.839, p = 0.003). However, the magnitude of the difference between cement line and osteonal calcium content decreased with increased osteonal calcium content (r = -0.709, p < 0.001), suggesting diverging mineralization dynamics in these osseous entities. The number of mineralized osteocyte lacunae per osteon bone area correlated positively with both osteonal and cement line calcium content (p < 0.01). The degree of mineralization of cement lines may represent another tissue-age related phenomenon, given that it strongly relates to the osteonal mineralization level. Understanding of the cement lines' mineralization and their changes in aging and disease states is important for predicting crack propagation pathways and fracture resistance mechanisms in human cortical bone.

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.

Micromechanical modeling of elastic properties of cortical bone accounting for anisotropy of dense tissue.

The paper analyzes the connection between microstructure of the osteonal cortical bone and its overall elastic properties. The existing models either neglect anisotropy of the dense tissue or simplify cortical bone microstructure (accounting for Haversian canals only). These simplifications (related mostly to insufficient mathematical apparatus) complicate quantitative analysis of the effect of microstructural changes - produced by age, microgravity, or some diseases - on the overall mechanical performance of cortical bone. The present analysis fills this gap; it accounts for anisotropy of the dense tissue and uses realistic model of the porous microstructure. The approach is based on recent results of Sevostianov et al. (2005) and Saadat et al. (2012) on inhomogeneities in a transversely-isotropic material. Bone's microstructure is modeled according to books of Martin and Burr (1989), Currey (2002), and Fung (1993) and includes four main families of pores. The calculated elastic constants for porous cortical bone are in agreement with available experimental data. The influence of each of the pore types on the overall moduli is examined.

Numerical modelling of the mechanical behaviour of an osteon with microcracks.

In this work, we present two strategies for the numerical modelling of microcracks and damage within an osteon. A numerical model of a single osteon under compressive diametral load is developed, including lamellae organized concentrically around the haversian canal and the presence of lacunae. Elastic properties have been estimated from micromechanical models that consider the mineralized collagen fibrils reinforced with hydroxyapatite crystals and the dominating orientation of the fibrils in each lamella. Microcracks are simulated through the node release technique, enabling propagation along the lamellae interfaces by application of failure criteria initially conceived for composite materials, in particular the Brewer and Lagacé criterion for delamination. A second approach is also presented, which is based on the progressive degradation of the stiffness at the element level as the damage increases. Both strategies are discussed, showing a good agreement with experimental evidence reported by other authors. It is concluded that interlaminar shear stresses are the main cause of failure of an osteon under compressive diametral load.

The osteoplastic effectiveness of the implants made of mesh titanium nickelide constructs.

The purpose of the work was to study the features of reparative osteogenesis for filling the defect of tubular bone under implantation of mesh titanium nickelide constructs. Tibial fenestrated defect was modeled experimentally in 30 Wistar pubertal rats, followed by implant intramedullary insertion. The techniques of radiography, scanning electron microscopy and X-ray electron probe microanalysis were used. The mesh implant of titanium nickelide has been established to possess biocompatibility, osteoconductive and osteoinductive properties, the zone of osteogenesis and angiogenesis is created around it, bone cover is formed. Osteointegration of the implant occurs early, by 7 days after surgery, and by 30 days after surgery organotypical re-modelling of the regenerated bone takes place, as well as the defect is filled with lamellar bone tissue by the type of bone wound primary adhesion. By 30 days after surgery mineral content of the regenerated bone tissue approximates to the composition of intact cortex mineral phase.

A bone remodelling model including the effect of damage on the steering of BMUs.

Bone remodelling in cortical bone is performed by the so-called basic multicellular units (BMUs), which produce osteons after completing the remodelling sequence. Burger et al. (2003) hypothesized that BMUs follow the direction of the prevalent local stress in the bone. More recently, Martin (2007) has shown that BMUs must be somehow guided by microstructural damage as well. The interaction of both variables, strain and damage, in the guidance of BMUs has been incorporated into a bone remodelling model for cortical bone. This model accounts for variations in porosity, anisotropy and damage level. The bone remodelling model has been applied to a finite element model of the diaphysis of a human femur. The trajectories of the BMUs have been analysed throughout the diaphysis and compared with the orientation of osteons measured experimentally. Some interesting observations, like the typical fan arrangement of osteons near the periosteum, can be explained with the proposed remodelling model. Moreover, the efficiency of BMUs in damage repairing has been shown to be greater if BMUs are guided by damage.

Cement lines and interlamellar areas in compact bone as strain amplifiers - contributors to elasticity, fracture toughness and mechanotransduction.

Bone is multi-scale hierarchical composite material making the prediction of fragility, as well as pinning it to a certain cause, complicated. For proper mechanical simulation and reflection of bone properties in models, microscopic structural features of bone tissue need to be included. This study sets out to gain a mechanistic insight into the role of various microstructural features of bone tissue in particular cement lines and interlamellar areas. Further the hypothesis that compliant interlamellar areas and cement lines within osteonal bone act as strain amplifiers was explored. To this end, a series of experimentally-based micromechanical finite element models of bovine osteonal bone were developed. Different levels of detail for the bone microstructure were considered and combined with the results of physical three-point bending tests and an analytical composite model of a single osteon. The objective was to examine local and global effects of interface structures. The geometrical and microstructural characteristics of the bone samples were derived from microscopy imaging. Parametric finite element studies were conducted to determine optimal values of the elastic modulus of interstitial bone and interlamellar areas. The average isotropic elastic modulus of interfaces suggested in this study is 88.5MPa. Based on the modelling results, it is shown that interfaces are areas of accumulated strain in bone and are likely to act as potential paths for crack propagation. The strain amplification capability of interface structures in the order of 10 predicted by the models suggests a new explanation for the levels of strain required in bone homoeostasis for maintenance and adaptation.

Evolutionary patterns of bone histology and bone compactness in xenarthran mammal long bones.

Bone microstructure reflects physiological characteristics and has been shown to contain phylogenetic and ecological signals. Although mammalian long bone histology is receiving increasing attention, systematic examination of the main clades has not yet been performed. Here we describe the long bone microstructure of Xenarthra based on thin sections representing twenty-two species. Additionally, patterns in bone compactness of humeri and femora are investigated. The primary bone tissue of xenarthran long bones is composed of a mixture of woven, parallel-fibered and lamellar bone. The vascular canals have a longitudinal, reticular or radial orientation and are mostly arranged in an irregular manner. Concentric rows of vascular canals and laminar organization of the tissue are only found in anteater bones. The long bones of adult specimens are marked by dense Haversian bone, a feature that has been noted for most groups of mammals. In the long bones of armadillos, secondary osteons have an oblique orientation within the three-dimensional bone tissue, thus resulting in their irregular shape when the bones are sectioned transversely. Secondary remodeling is generally more extensive in large taxa than in small taxa, and this could be caused by increased loading. Lines of arrested growth are assumed to be present in all specimens, but they are restricted to the outermost layer in bones of armadillos and are often masked by secondary remodeling in large taxa. Parameters of bone compactness show a pattern in the femur that separates Cingulata and Pilosa (Folivora and Vermilingua), with cingulates having a lower compactness than pilosans. In addition, cingulates show an allometric relationship between humeral and femoral bone compactness.

Architecture of the osteocyte network correlates with bone material quality.

In biological tissues such as bone, cell function and activity crucially depend on the physical properties of the extracellular matrix which the cells synthesize and condition. During bone formation and remodeling, osteoblasts get embedded into the matrix they deposit and differentiate to osteocytes. These cells form a dense network throughout the entire bone material. Osteocytes are known to orchestrate bone remodeling. However, the precise role of osteocytes during mineral homeostasis and their potential influence on bone material quality remains unclear. To understand the mutual influence of osteocytes and extracellular matrix, it is crucial to reveal their network organization in relation to the properties of their surrounding material. Here we visualize and topologically quantify the osteocyte network in mineralized bone sections with confocal laser scanning microscopy. At the same region of the sample, synchrotron small-angle X-ray scattering is used to determine nanoscopic bone mineral particle size and arrangement relative to the cell network. Major findings are that most of the mineral particles reside within less than a micrometer from the nearest cell network channel and that mineral particle characteristics depend on the distance from the cell network. The architecture of the network reveals optimization with respect to transport costs between cells and to blood vessels. In conclusion, these findings quantitatively show that the osteocyte network provides access to a huge mineral reservoir in bone due to its dense organization. The observed correlation between the architecture of osteocyte networks and bone material properties supports the hypothesis that osteocytes interact with their mineralized vicinity and thus, participate in bone mineral homeostasis.

Changes to the cell, tissue and architecture levels in cranial suture synostosis reveal a problem of timing in bone development.

Premature fusion of cranial sutures is a common problem with an incidence of 3-5 per 10,000 live births. Despite progress in understanding molecular/genetic factors affecting suture function, the complex process of premature fusion is still poorly understood. In the present study, corresponding excised segments of nine patent and nine prematurely fused sagittal sutures from infants (age range 3-7 months) with a special emphasis on their hierarchical structural configuration were compared. Cell, tissue and architecture characteristics were analysed by transmitted and polarised light microscopy, 2D-histomorphometry, backscattered electron microscopy and energy-dispersive-x-ray analyses. Apart from wider sutural gaps, patent sutures showed histologically increased new bone formation compared to reduced new bone formation and osseous edges with a more mature structure in the fused portions of the sutures. This pattern was accompanied by a lower osteocyte lacunar density and a higher number of evenly mineralised osteons, reflecting pronounced lamellar bone characteristics along the prematurely fused sutures. In contrast, increases in osteocyte lacunar number and size accompanied by mineralisation heterogeneity and randomly oriented collagen fibres predominantly signified woven bone characteristics in patent, still growing suture segments. The already established woven-to-lamellar bone transition provides evidence of advanced bone development in synostotic sutures. Since structural and compositional features of prematurely fused sutures did not show signs of pathological/defective ossification processes, this supports the theory of a normal ossification process in suture synostosis - just locally commencing too early. These histomorphological findings may provide the basis for a better understanding of the pathomechanism of craniosynostosis, and for future strategies to predict suture fusion and to determine surgical intervention.

A model of osteoblast-osteocyte kinetics in the development of secondary osteons in rabbits.

The kinetics of osteogenic cells within secondary osteons have been examined within a 2-D model. The linear osteoblast density of the osteons and the osteocyte lacunae density were compared with other endosteal lamellar systems of different geometries. The cell density was significantly greater in the endosteal appositional zone and was always flatter than the central osteonal canals. Fully structured osteons compared with early structuring (cutting cones) did not show any significant differences in density. The osteoblast density may remain constant because some of them leave the row and become embedded within matrix. The overall shape of the Haversian system represented a geometrical restraint and it was thought to be related to osteoblast-osteocyte transformation. To test this hypothesis of an early differentiation and recruitment of the osteoblast pool which completes the lamellar structure of the osteon, the number and density of osteoblasts and osteocyte lacunae were evaluated. In the central canal area, the mean osteoblast linear density and the osteocyte lacunae planar density were not significantly different among sub-classes (with the exclusion of the osteocyte lacunae of the 300-1000 µm(2) sub-class). The mean number of osteoblasts compared with osteocyte lacunae resulted in significantly higher numbers in the two sub-classes, no significant difference was seen in the two middle sub-classes with the larger canals, and there were significantly lower levels in the smallest central canal sub-class. The TUNEL technique was used to identify the morphological features of apoptosis within osteoblasts. It was found that apoptosis occurred during the late phase of osteon formation but not in osteocytes. This suggests a regulatory role of apoptosis in balancing the osteoblast-osteocyte equilibrium within secondary osteon development. The position of the osteocytic lacunae did not correlate with the lamellar pattern and the lacunae density in osteonal radial sectors was not significantly different. These findings support the hypothesis of an early differentiation of the osteoblast pool and the independence of the fibrillar lamellation from osteoblast-osteocyte transformation.

Bone laminarity in the avian forelimb skeleton and its relationship to flight mode: testing functional interpretations.

Wing bone histology in three species of birds was characterized in order to test hypotheses related to the relationship between skeletal microstructure and inferred wing loading during flight. Data on the degree of laminarity (the proportion of circular vascular canals) and the occurrence of secondary osteons were obtained from three species that utilize different primary flight modes: the Double-crested cormorant, a continuous flapper; the Brown pelican, a static soarer; and the Laysan albatross, a dynamic soarer. Laminarity indices were calculated for four quadrants for each of the three main wing elements. Ulnae and carpometacarpi were predicted to exhibit quadrant specific patterns of laminarity due to hypothesized differences in locally applied loads related to the attachment of flight feathers. However, few differences among the quadrants were identified. No significant differences were identified among the three elements, which is notable as different bones are likely experiencing different loading conditions. These results do not support the concept of bone functional adaptation in the primary structure of the wing elements. Significant differences in laminarity were found among the three primary flight modes. The dynamic soaring birds exhibited significantly lower laminarity than the flapping and static soaring birds. These results support the proposed hypothesis that laminarity is an adaptation for resisting torsional loading. This may be explained by overall wing shape: whereas dynamic soaring birds have long slender wings, flappers and static soaring birds have broader wings with a larger wing chord that would necessarily impart a higher torsional moment on the feather-bearing bones.

Bisphosphonate binding affinity affects drug distribution in both intracortical and trabecular bone of rabbits.

Differences in the binding affinities of bisphosphonates for bone mineral have been proposed to determine their localizations and duration of action within bone. The main objective of this study was to test the hypothesis that mineral binding affinity affects bisphosphonate distribution at the basic multicellular unit (BMU) level within both cortical and cancellous bone. To accomplish this objective, skeletally mature female rabbits (n = 8) were injected simultaneously with both low- and high-affinity bisphosphonate analogs bound to different fluorophores. Skeletal distribution was assessed in the rib, tibia, and vertebra using confocal microscopy. The staining intensity ratio between osteocytes contained within the cement line of newly formed rib osteons or within the reversal line of hemiosteons in vertebral trabeculae compared to osteocytes outside the cement/reversal line was greater for the high-affinity compared to the low-affinity compound. This indicates that the low-affinity compound distributes more equally across the cement/reversal line compared to a high-affinity compound, which concentrates mostly near surfaces. These data, from an animal model that undergoes intracortical remodeling similar to humans, demonstrate that the affinity of bisphosphonates for the bone determines the reach of the drugs in both cortical and cancellous bone.

Osteonic organization of limb bones in mammals, including humans, and birds: a preliminary study.

As it is well known, bone tissue is characterized by a calcified extracellular matrix which makes this tissue suitable to support the body and protect the inner organs. Lamellar bone tissue is organized in lamellae, 3-7 microm in thickness, and arranged concentrically around vascular channels: the basic structure in this type of organization is called Haversian system or osteon and the diameter of osteons depends on the number of lamellae. Shape and regional density of osteons are related to the bone segment and the specific functional requirements to meet. Aim of this study is to correlate the compact bone tissue microstructure in various classes of mammals, including humans, and birds in order to find an adequate identification key. The results of our study show that in bone tissue samples from various classes of mammals, including humans, and birds the osteonic structure shows peculiar features, often depending on the rate of bone remodelling, different in different animal species. We conclude that a careful microscopic analysis of bone tissue and the characterization of distinctive osteonic features could give a major contribution to forensic medicine to obtain a more reliable recognition of bone findings.

Analysis of the effect of osteon diameter on the potential relationship of osteocyte lacuna density and osteon wall thickness.

An important hypothesis is that the degree of infilling of secondary osteons (Haversian systems) is controlled by the inhibitory effect of osteocytes on osteoblasts, which might be mediated by sclerostin (a glycoprotein produced by osteocytes). Consequently, this inhibition could be proportional to cell number: relatively greater repression is exerted by progressively greater osteocyte density (increased osteocytes correlate with thinner osteon walls). This hypothesis has been examined, but only weakly supported, in sheep ulnae. We looked for this inverse relationship between osteon wall thickness (On.W.Th) and osteocyte lacuna density (Ot.Lc.N/B.Ar) in small and large osteons in human ribs, calcanei of sheep, deer, elk, and horses, and radii and third metacarpals of horses. Analyses involved: (1) all osteons, (2) smaller osteons, either ≤150 µm diameter or less than or equal to the mean diameter, and (3) larger osteons (>mean diameter). Significant, but weak, correlations between Ot.Lc.N/B.Ar and On.W.Th/On.Dm (On.Dm = osteon diameter) were found when considering all osteons in limb bones (r values -0.16 to -0.40, P < 0.01; resembling previous results in sheep ulnae: r = -0.39, P < 0.0001). In larger osteons, these relationships were either not significant (five/seven bone types) or very weak (two/seven bone types). In ribs, a negative relationship was only found in smaller osteons (r = -0.228, P < 0.01); this inverse relationship in smaller osteons did not occur in elk calcanei. These results do not provide clear or consistent support for the hypothesized inverse relationship. However, correlation analyses may fail to detect osteocyte-based repression of infilling if the signal is spatially nonuniform (e.g., increased near the central canal).

Imaging the 3D structure of secondary osteons in human cortical bone using phase-retrieval tomography.

By applying a phase-retrieval step before carrying out standard filtered back-projection reconstructions in tomographic imaging, we were able to resolve structures with small differences in density within a densely absorbing sample. This phase-retrieval tomography is particularly suited for the three-dimensional segmentation of secondary osteons (roughly cylindrical structures) which are superimposed upon an existing cortical bone structure through the process of turnover known as remodelling. The resulting images make possible the analysis of the secondary osteon structure and the relationship between an osteon and the surrounding tissue. Our observations have revealed many different and complex 3D structures of osteons that could not be studied using previous methods. This work was carried out using a laboratory-based x-ray source, which makes obtaining these sorts of images readily accessible.

Analysis of osteon morphotype scoring schemes for interpreting load history: evaluation in the chimpanzee femur.

Osteon morphotype scores (MTSs) allow for quantification of mechanically important collagen/lamellar variations between secondary osteons when viewed in circularly polarized ight (CPL). We recently modified the 6-point MTS method of Martin et al. (Martin RB, Gibson VA, Stover SM, Gibeling JC, Griffin LV (1996a) Osteonal structure in the equine third metacarpus. Bone 19, 165-71) and reported superiority of this modified method in correlating with 'tension' and 'compression' cortices of both chimpanzee proximal femoral diaphyses and diaphyses of other non-anthropoid bones that are loaded in habitual bending (Skedros et al. 2009, 2011). In these studies, the 'tension' and 'compression' cortices differed significantly in predominant collagen fiber orientation (CFO) based on weighted-mean gray levels (CFO/WMGLs) in CPL images. In chimpanzee femora, however, some osteons were difficult to score with the 6-point method; namely, 'hybrids' with peripherally bright 'hoops' and variability in alternating rings within the osteon wall. We hypothesized that some of these hybrids would be more prevalent in regions subject to torsion than bending. In this perspective the present study was aimed at expanding our 6-point scoring method (S-6-MTS) into two 12-point methods with six additional morphotypes that considered these hybrids. Three- and 4-point methods were also evaluated. We hypothesized that at least one of these other methods would out-perform the S-6-MTS in terms of accuracy, reliability, and interpreting torsion vs. bending load histories. Osteon morphotypes were quantified in CPL images from transverse sections of eight adult chimpanzee femora (neck, proximal diaphysis, mid-diaphysis), where the mid-diaphysis and base- and mid-neck locations have relatively more complex loading (e.g. torsion + bending) than the proximal diaphysis, where bending predominates. Correlation coefficients between CFO/WMGL and MTSs showed that the S-6-MTS method was either stronger or equivalent to the 12-point methods, and typically stronger than the 3- and 4-point methods for all load environments. In nearly all instances the S-6-MTS is more reliable and accurate when it is applied to cases where interpreting load history requires distinguishing habitual bending from torsion. Consequently, in studies of osteonal adaptations for these load histories the 3- and 4-point methods are not stronger correlates, and the extra time required to assign additional scores in the 12-point methods is both unnecessary and can be highly unreliable.