Morphometric analysis of osteonal architecture in bones from healthy young human male subjects using scanning electron microscopy

The shape and structure of bones is a topic that has been studied for a long time by morphologists and biologists with the goal of explaining the laws governing their development, aging and pathology. The osteonal architecture of tibial and femoral mid‐diaphyses was examined morphometrically with scanning electron microscopy in four healthy young male subjects. In transverse sections of the mid‐diaphysis, the total area of the anterior, posterior, lateral and medial cortex sectors was measured and analysed for osteonal parameters including osteon number and density, osteon total and bone area and vascular space area. Osteons were grouped into four classes including cutting heads (A), transversely cut osteons (B), longitudinally cut osteons (C) and sealed osteons (D). The morphometric parameters were compared between the inner (endosteal) and outer (periosteal) half of the cortex. Of 5927 examined osteons, 24.4% cutting heads, 71.1% transversely cut osteons, 2.3% longitudinally cut osteons and 2.2% sealed osteons were found. The interosteonic bone (measured as the area in a lamellar system that has lost contact with its own central canal) corresponded to 51.2% of the endosteal and 52.4% of the periosteal half‐cortex. The mean number of class A cutting heads and class B osteons was significantly higher in the periosteal than in the endosteal half‐cortex (P < 0.001 and P < 0.05, respectively), whereas there was no significant difference in density. The mean osteon total area, osteon bone area and vascular space area of both classes A and B were significantly higher (P < 0.001 for all three parameters) in the endosteal than in the periosteal half‐cortex. The significant differences between the two layers of the cortex suggest that the osteoclast activity is distributed throughout the whole cortical thickness, with more numerous excavations in the external layer, but larger resorption lacunae closer to the marrow canal. A randomly selected population of 109 intact class B osteons was examined at higher magnification (350×) to count osteocyte lacuna and to analyse their relationship with osteon size parameters. The distribution frequency of the mean number of osteocyte lacunae increased with the increment in the sub‐classes of osteon bone area, whereas the density did not show significant differences. The number of osteocyte lacunae had a direct correlation with the osteon bone area and the mean osteon wall thickness, as well as the mean number of lamellae. The osteocyte lacunae density showed an inverse relationship. These data suggest a biological regulation of osteoblast activity with a limit to the volume of matrix produced by each cell and proportionality with the number of available cells in the space of the cutting cone (total osteon area). The collected data can be useful as a set of control parameters in healthy human bone for studies on bone aging and metabolic bone diseases.     

Osteoblast‐osteocyte transformation. A SEM densitometric analysis of endosteal apposition in rabbit femur

Transformation of osteoblasts into osteocytes is marked by changes in volume and cell shape. The reduction of volume and the entrapment process are correlated with the synthesis activity of the cell which decreases consequently. This transformation process has been extensively investigated by transmission electron microscopy (TEM) but no data have yet been published regarding osteoblast-osteocyte dynamic histomorphometry. Scanning electron microscope (SEM) densitometric analysis was carried out to determine the osteoblast and open osteocyte lacunae density in corresponding areas of a rabbit femur endosteal surface. The lining cell density was 4900.1 ± 30.03 n mm⁻², the one of open osteocyte lacunae 72.89 ± 22.55 n mm⁻². This corresponds to an index of entrapment of one cell every 67.23 osteoblasts (approximated by defect). The entrapment sequence begins with flattening of the osteoblast and spreading of equatorial processes. At first these are covered by the new apposed matrix and then also the whole cellular body of the osteocyte undergoing entrapment. The dorsal aspect of the cell membrane suggests that closure of the osteocyte lacuna may be partially carried out by the same osteoblast-osteocyte which developed a dorsal secretory territory. A significant proportion of the endosteal surface was analysed by SEM, without observing any evidence of osteoblast mitotic figures. This indicates that recruitment of the pool of osteogenic cells in cortical bone lamellar systems occurs prior to the entrapment process. No further additions occurred once osteoblasts were positioned on the bone surface and began lamellar apposition. The number of active osteoblasts on the endosteal surface exceeded that of the cells which become incorporated as osteocytes (whose number was indicated by the number of osteocyte lacunae). Therefore such a balance must be equilibrated by the osteoblasts'' transformation in resting lining cells or by apoptosis. The current work characterised osteoblast shape changes throughout the entrapment process, allowing approximate calculation of an osteoblast entrapment index in the rabbit endosteal cortex.     

Accumulation of type VI collagen in the primary osteon of the rat femur during postnatal development

In rodents, the long bone diaphysis is expanded by forming primary osteons at the periosteal surface of the cortical bone. This ossification process is thought to be regulated by the microenvironment in the periosteum. Type VI collagen (Col VI), a component of the extracellular matrix (ECM) in the periosteum, is involved in osteoblast differentiation at early stages. In several cell types, Col VI interacts with NG2 on the cytoplasmic membrane to promote cell proliferation, spreading and motility. However, the detailed functions of Col VI and NG2 in the ossification process in the periosteum are still under investigation. In this study, to clarify the relationship between localization of Col VI and formation of the primary osteon, we examined the distribution of Col VI and osteoblast lineages expressing NG2 in the periosteum of rat femoral diaphysis during postnatal growing periods by immunohistochemistry. Primary osteons enclosing the osteonal cavity were clearly identified in the cortical bone from 2 weeks old. The size of the osteonal cavities decreased from the outer to the inner region of the cortical bone. In addition, the osteonal cavities of newly formed primary osteons at the outermost region started to decrease in size after rats reached the age of 4 weeks. Immunohistochemistry revealed concentrated localization of Col VI in the ECM in the osteonal cavity. Col VI-immunoreactive areas were reduced and they disappeared as the osteonal cavities became smaller from the outer to the inner region. In the osteonal cavities of the outer cortical regions, Runx2-immunoreactive spindle-shaped cells and mature osteoblasts were detected in Col VI-immunoreactive areas. The numbers of Runx2-immunoreactive cells were significantly higher in the osteonal cavities than in the osteogenic layers from 2 to 4 weeks. Most of these Runx2-immunoreactive cells showed NG2-immunoreactivity. Furthermore, PCNA-immunoreactivity was detected in the Runx2-immunoreactive spindle cells in the osteonal cavities. These results indicate that Col VI provides a characteristic microenvironment in the osteonal cavity of the primary osteon, and that differentiation and proliferation of the osteoblast lineage occur in the Col VI-immunoreactive area. Interaction of Col VI and NG2 may be involved in the structural organization of the primary osteon by regulating osteoblast lineages.     

Morphometry and Patterns of Lamellar Bone in Human Haversian Systems

The lamellar architecture of secondary osteons (Haversian systems) has been studied with scanning electron microscopy (SEM) in transverse sections of human cortical bone. Na₃PO₄ etching was used to improve the resolution of the interface between neighboring lamellae and the precision of measurements. These technical improvements permitted testing of earlier morphometry assumptions concerning lamellar thickness while revealing the existence of different lamellar patterns. The mean lamellar thickness was 9.0 ± 2.13 μm, thicker and with a wider range of variation with respect to earlier measurements. The number of lamellae showed a direct correlation with the lamellar bone area, and their thickness had a random distribution for osteonal size classes. The circular, concentrical pattern was the more frequently observed, but spiral and crescent‐moon‐shaped lamellae were also documented. Selected osteons were examined by either SEM or SEM combined with polarized light microscopy allowing comparisons of corresponding sectors of the osteon. The bright bands observed with polarized light corresponded to the grooves observed in etched sections by SEM. The dark bands corresponded to the lamellar surface with the cut fibrils oriented approximately longitudinally along the central canal axis. However, lamellae with large and blurred bright bands could be observed, which did not correspond to a groove observed by SEM. These findings are in contrast with the assumption that all the fibril layers within a lamella are oriented along a constant and unchangeable angle. The different lamellar patterns may be explained by the synchronous or staggered recruitment and activation of osteoblasts committed to the osteon's completion. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.     

From bone lining cell to osteocyte--an SEM study

We describe the SEM appearance of the rat endosteal bone lining cell ( BLC ) population, and the sequence of morphological changes of these cells as they self-incorporate into unmineralized bone matrix (osteoid), establish intercellular connections, and construct lacunae. The osteoblast/nascent osteocyte series was progressively unsheathed by gentle digestion of the osteoid with 0.25% collagenase. The osteoblasts which leave the polygonally packed BLC compartment rapidly develop numerous complexly branched processes that contact the processes elaborated by previous generations of maturing and mature osteocytes. As osteoblasts mature and approach the mineralization front, they appear to lose processes. The mature cells begin to form osteocyte lacunae by depositing an asymmetric perimeter of woven collagen fibrils, such that as the cells roof-over, the lacunae appear as pocketlike constructions. The collagen fibrils on the perilacunar matrix are oriented in a tangential or circular pattern, while those in the more distal matrix are arranged in a parallel pattern. With the completion of a lacuna, its wall appears to mineralize quickly, for lacunae could be recognized only when they are forming.     

From bone lining cell to osteocyte—an SEM study

We describe the SEM appearance of the rat endosteal bone lining cell (BLC) population, and the sequence of morphological changes of these cells as they self-incorporate into unmineralized bone matrix (osteoid), establish intercellular connections, and construct lacunae. The osteoblast/nascent osteocyte series was progressively unsheathed by gentle digestion of the osteoid with 0.25% collagenase. The osteoblasts which leave the polygonally packed BLC compartment rapidly develop numerous complexly branched processes that contact the processes elaborated by previous generations of maturing and mature osteocytes. As osteoblasts mature and approach the mineralization front, they appear to lose processes. The mature cells begin to form osteocyte lacunae by depositing an asymmetric perimeter of woven collagen fibrils, such that as the cells roof-over, the lacunae appear as pocketlike constructions. The collagen fibrils on the perilacunar matrix are oriented in a tangential or circular pattern, while those in the more distal matrix are arranged in a parallel pattern. With the completion of a lacuna, its wall appears to mineralize quickly, for lacunae could be recognized only when they are forming.     

Multi-level characterization of human femoral cortices and their underlying osteocyte network reveal trends in quality of young,aged, osteoporotic and antiresorptive-treated bone

Characterization of bone's hierarchical structure in aging, disease and treatment conditions is imperative to understand the architectural and compositional modifications to the material and its mechanical integrity. Here, cortical bone sections from 30 female proximal femurs – a frequent fracture site – were rigorously assessed to characterize the osteocyte lacunar network, osteon density and patterns of bone matrix mineralization by backscatter-electron imaging and Fourier-transform infrared spectroscopy in relation to mechanical properties obtained by reference-point indentation. We show that young, healthy bone revealed the highest resistance to mechanical loading (indentation) along with higher mineralization and preserved osteocyte-lacunar characteristics. In contrast, aging and osteoporosis significantly alter bone material properties, where impairment of the osteocyte-lacunar network was evident through accumulation of hypermineralized osteocyte lacunae with aging and even more in osteoporosis, highlighting increased osteocyte apoptosis and reduced mechanical competence. But antiresorptive treatment led to fewer mineralized lacunae and fewer but larger osteons signifying rejuvenated bone. In summary, multiple structural and compositional changes to the bone material were identified leading to decay or maintenance of bone quality in disease, health and treatment conditions. Clearly, antiresorptive treatment reflected favorable effects on the multifunctional osteocytic cells that are a prerequisite for bone's structural, metabolic and mechanosensory integrity.     

Super-osteons (remodeling clusters) in the cortex of the femoral shaft: influence of age and gender.

Previous studies of cortical remodeling in the fractured femoral neck indicated that the merging of spatially clustered remodeling osteons could result in the formation of deleteriously large cavities associated with femoral neck fracture. This study aimed to identify whether remodeling osteons in the femoral shaft were also clustered and to assess the influence of age and gender. Microradiographic images of femoral mid-shaft cross-sections from 66 subjects over 21 years of age were analyzed to determine the number, size and location of all Haversian canals. Those most recently remodeled were identified using an edge-detection algorithm highlighting the most marked differential gradients in grey levels. Cluster analysis (JMP software) of these osteons identified the proportion of recently remodeled osteons that were within 0.75 mm clusters. As in the femoral neck, remodeling osteons were significantly more clustered than could occur by chance (real, 59.4%; random, 39.4%; P < 0.0001). The density of these clusters (number/mm(2)) was not significantly associated with subject age or gender but was greatest near the periosteum and decreased toward the marrow cavity (periosteal 0.043 +/- 0.004; mid-cortex 0.028 +/- 0.003; endosteal 0.017 +/- 0.002). Cortical porosity increased with age. The presence of giant canals (diameter >385 microm) was inversely related to the presence of clusters (R(2) = 0.237, P < 0.0001). This data suggest that remodeling osteons tend to be spatially colocalized in the shaft as they are in the neck of the femur and their presence is independent of age or gender. We propose that these remodeling clusters be termed super-osteons. The negative relationship between super-osteons and giant canals raises the intriguing possibility that loss of the control of remodeling depth results in the merging of osteonal systems to form deleteriously large cortical cavities with a marked reduction in bone strength.     

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). Anat Rec,, 2011. © 2011 Wiley‐Liss, Inc.     

Histomorphological and geometric properties of human femoral cortex in individuals over 50: Implications for histomorphological determination of age-at-death

Cortical area, area moment of inertia, and polar moment of inertia were determined from the midshafts of a series of 62 femurs (34 female and 28 male) from a U. S. white population, ages 51–95. The density of osteons and osteon fragments (per mm²) was also determined. Neither osteon nor osteon fragment density was significantly correlated with age. These variables were correlated, however, with normalized cortical and endosteal areas, normalized area moment of inertia, and polar moment of inertia. Osteon fragment numbers alone are not highly correlated with cross-sectional properties. This research suggests that osteon density and osteon fragment density are significantly related to cortical mass and distribution in older people, but are not a direct function of age in persons over 50 years of age. Histological age estimates in older individuals must, therefore, be used with extreme caution. © 1994 Wiley-Liss, Inc.     

Super‐osteons (remodeling clusters) in the cortex of the femoral shaft: Influence of age and gender

Previous studies of cortical remodeling in the fractured femoral neck indicated that the merging of spatially clustered remodeling osteons could result in the formation of deleteriously large cavities associated with femoral neck fracture. This study aimed to identify whether remodeling osteons in the femoral shaft were also clustered and to assess the influence of age and gender. Microradiographic images of femoral mid‐shaft cross‐sections from 66 subjects over 21 years of age were analyzed to determine the number, size and location of all Haversian canals. Those most recently remodeled were identified using an edge‐detection algorithm highlighting the most marked differential gradients in grey levels. Cluster analysis (JMP software) of these osteons identified the proportion of recently remodeled osteons that were within 0.75mm clusters. As in the femoral neck, remodeling osteons were significantly more clustered than could occur by chance (real, 59.4%; random, 39.4%; P < 0.0001). The density of these clusters (number/mm²) was not significantly associated with subject age or gender but was greatest near the periosteum and decreased toward the marrow cavity (periosteal 0.043 ± 0.004; mid‐cortex 0.028 ± 0.003; endosteal 0.017 ± 0.002). Cortical porosity increased with age. The presence of giant canals (diameter >385μm) was inversely related to the presence of clusters (R² = 0.237, P < 0.0001). This data suggest that remodeling osteons tend to be spatially colocalized in the shaft as they are in the neck of the femur and their presence is independent of age or gender. We propose that these remodeling clusters be termed super‐osteons. The negative relationship between super‐osteons and giant canals raises the intriguing possibility that loss of the control of remodeling depth results in the merging of osteonal systems to form deleteriously large cortical cavities with a marked reduction in bone strength. Anat Rec 264:378–386, 2001. © 2001 Wiley‐Liss, Inc.     

Histomorphometric study on the osteocyte lacuno-canalicular network in animals of different species. II. Parallel-fibered and lamellar bones

The shape, size and density of osteocyte lacunae in parallel-fibered and lamellar bone were histomorphometrically analyzed in relation to the organization of the collagen fiber texture and the animal species (frog, sheep, dog, bovine, horse and man). The following parameters were measured under the light microscope (LM) by a computer-assisted image analyzer: 1) shape, size and distribution of osteocyte lacunae; 2) osteocyte lacuno-canalicular density. In close agreement with our previous studies, which includes woven bone, it resulted that in all animals (even in frog) osteocyte lacunae have a rounded globous shape in woven bone and an oval shape in both parallel-fibered and lamellar bone; in the latter, however, they are more flattened, only located in loose lamellae and thus regularly distributed in rows. Osteocyte lacunar density is higher in woven-fibered, intermediate in parallel-fibered and lower in lamellar bone, whereas no correlation seems to exist with the animal species. In conclusion, these results suggest that osteocyte shape, size and density seem to depend mainly on collagen fiber texture rather than on the animal species. The role of osteocyte-recruitment on the spatial organization of collagen fibers in bone tissues is discussed.     

The fibrillar organisation of the osteon and cellular aspects of its development

The collagen architecture of secondary osteons was studied with scanning electron microscopy (SEM) employing the fractured cortex technique and osmic maceration. Fibrillar orientation and the change in their direction in sequential lamellae was documented where lamellar formation was ongoing, as well as in resorption pits where osteoclasts had exposed the collagen organisation of the underlying layers. Applying an adaptive stereo matching technique, the mean thickness of matrix layers removed by osteoclasts was 1.36 ± 0.45 μm. It was also documented that osteoclasts do not attack the cellular membrane of the exposed osteocytes. The mean linear osteoblast density in fractured hemicanals was assessed with SEM and no significant differences were observed comparing larger with smaller central canal osteons. These findings suggested a balance between the differentiated osteoblasts that have aligned on the surface of the cutting cone and those that are transformed into osteocytes, because the canal surface is progressively reduced as the lamellar apposition advances.     

An electron microscopic study on the presence of proteoglycans in the mineralized matrix of rat and human compact lamellar bone

The presence of proteoglycans (PGs) was studied in compact lamellar rat and human bone at the electron microscopic level. With the cationic dye cuprolinic blue (CBI), PGs could be demonstrated in the mineralized bone matrix. The amounts of PGs appeared to be equal in the different lamellae and osteons. More CBI-positive material was found in the outermost lamella of the cortex, in the perilacunar matrix around the osteocyte lacunae, and around the canaliculi. Enzyme digestion with chondroitinase ABC demonstrated that the CBI-positive rods consisted of PGs. These observations amplify biochemical studies in which PGs have been isolated from the mineralized bone matrix. The presence of CBl-positive rods in the mineralized matrix suggest that PGs do not have to be removed completely to make the matrix calcifiable.     

Histomorphometric assessment of Haversian canal and osteocyte lacunae in different-sized osteons in human rib

There is no detailed information available concerning the variations in bone, the Haversian canal, and osteocyte populations in different-sized osteons. In this study a total of 398 secondary osteons were measured in archived rib sections from nine white men (20-25 years old). The sections were stained with basic fuchsin. The parameters included the osteon area (On.Ar), Haversian canal area (HC.Ar) and perimeter (HC.Pm), bone area (B.Ar), and osteocyte lacunar number (Lc.N). From these primary measurements the following indices were deduced: 1) lacunar number per bone area (Lc.N/B.Ar) and per osteon (Lc.N/On); 2) the ratio between Haversian canal perimeter and bone area (HC.Pm/B.Ar); and 3) the fraction of Haversian canal area (HC.Ar/On.Ar) and its complement, the fraction of bone area (B.Ar/On.Ar). The results showed that the osteons varied greatly in size, but very little in the fraction of bone area. Regression analyses showed that HC.Ar, HC.Pm, and Lc.N/On were positively associated with On.Ar (P < 0.001 for all). A significant negative correlation was found between On.Ar and Lc.N/B.Ar (P < 0.05) and HC.Pm/B.Ar (P < 0.0001). HC.Ar and HC.Pm increased significantly with increasing Lc.N/On (both P < 0.0001) rather than Lc.N/B.Ar. Lc.N/B.Ar had a significant positive correlation with HC.Ar/On.Ar (P < 0.05) and HC.Pm/B.Ar (P < 0.01). We conclude that: 1) the size of the osteon is determined by the quantum of bone removed by osteoclasts, 2) the osteon is well designed for molecular exchange, and 3) a well designed osteon may be produced via the regulation of bone apposition by osteocytes during the process of osteon refilling.     

Collagen texture and osteocyte distribution in lamellar bone

A comparative scanning and transmission electron microscopy study was carried out on collagen fiber texture and osteocyte lacunae distribution in human lamellar bone. The results show that bony lamellae are not made up of parallel-arranged collagen fibers, as classically maintained. They are instead made up of highly interlaced fibers, and the lamellation appears to be due to the alternation of collagen-rich and collagen-poor layers, namely of dense and loose lamellae. The present study additionally shows that osteocyte lacunae are only located inside loose lamellae. Such structural organization of lamellar bone is briefly discussed in terms of bone biomechanics and osteogenesis.     

Principal stiffness orientation and degree of anisotropy of human osteons based on nanoindentation in three distinct planes

Haversian systems or 'osteons' are cylindrical structures, formed by bone lamellae, that make up the major part of human cortical bone. Despite their discovery centuries ago in 1691 by Clopton Havers, their mechanical properties are still poorly understood. The objective of this study is a detailed identification of the anisotropic elastic properties of the secondary osteon in the lamella plane. Additionally, the principal material orientation with respect to the osteon is assessed. Therefore a new nanoindentation method was developed which allows the measurement of indentation data in three distinct planes on a single osteon. All investigated osteons appeared to be anisotropic with a preferred stiffness alignment along the axial direction with a small average helical winding around the osteon axis. The mean degree of anisotropy was 1.75±0.36 and the mean helix angle was 10.3°±0.8°. These findings oppose two well established views of compact bone microstructure: first, the generally clear axial stiffness orientation contradicts a regular 'twisted plywood' collagen fibril orientation pattern in lamellar bone that would lead to a more isotropic behavior. Second, the class of transverse osteons were not observed from the mechanical point of view.     

Does 3D orientation account for variation in osteon morphology assessed by 2D histology?

The primary microstructural unit of cortical bone, the secondary osteon or Haversian system, is widely assumed to have a cylindrical shape. It is generally accepted that osteons are roughly circular in cross-section and deviations from circularity have been attributed to deviations from longitudinal orientation. To our knowledge this idealized geometric relationship, which assumes osteons are perfect cylinders, has not been rigorously explored. As such, we sought to explore two research questions: (i) Does the orientation of osteons in 3D explain variation in shapes visualized in 2D? (ii) Can differences in osteon 3D orientation explain previously reported age-related differences observed in their 2D cross-sectional shape (e.g. more circular shape and decreased area with age)? To address these questions we utilized a combination of 2D histology to identify osteon shape and superimposed micro-computed tomography data to assess osteon orientation in 3D based upon the osteonal canal. Shape was assessed by the inverse of Aspect Ratio (On.AspR⁻¹, based on a fitted ellipse) – which ranged from 0 (infinitely elongated shape) to 1 (perfectly circular). A sample (n = 27) of human female anterior femoral cortical bone samples from across the human lifespan (20–87 years) were included in the analysis, which involved 1418 osteons. The overall mean measure of On.AspR⁻¹ was 0.703 (1.42 Aspect Ratio). Mean osteon orientation was 79.1° (90° being longitudinal). While we anticipated a positive relation between orientation and On.AspR⁻¹, we found the opposite – a weak negative correlation (with more oblique 3D osteon alignment, the 2D shape became more circular as reflected by increased On.AspR⁻¹). When analysis of covariance was performed with age and orientation as covariates, the negative relation with orientation was replaced by a significant relation with age alone. This relation with age accounted for 41% of the variation of On.AspR⁻¹. The results revealed that osteons, on average, are not circular in cross-section and that 3D orientation cannot account for deviation from circular shape. Osteons thus are strictly speaking not cylinders, as they tend to have elliptical cross-sections. We observed that osteons did become less elliptical in cross-section with age independent of orientation – suggesting this is a real change in morphology.     

Poorly ordered bone as an endogenous scaffold for the deposition of highly oriented lamellar tissue in rapidly growing ovine bone

The mechanical properties of bone are known to depend on its structure at all length scales. In large animals, such as sheep, cortical bone grows very quickly and it is known that this occurs in 2 stages whereby a poorly ordered (mostly woven) bone structure is initially deposited and later augmented and partially replaced by parallel fibered and lamellar bone with much improved mechanical properties, often called primary osteons. Most interestingly, a similar sequence of events has also recently been observed during callus formation in a sheep osteotomy model. This has prompted the idea that fast intramembranous bone formation requires an intermediate step where bone with a lower degree of collagen orientation is deposited first as a substrate for osteoblasts to coordinate the synthesis of lamellar tissue. Since some osteoblasts become embedded in the mineralizing collagen matrix which they synthesize, the resulting osteocyte network is a direct image of the location of osteoblasts during bone formation. Using 3-dimensional imaging of osteocyte networks as well as tissue characterization by polarized light microscopy and backscattered electron imaging, we revisit the structure of growing plexiform (fibrolamellar) bone and callus in sheep. We show that bone deposited initially is based on osteocytes without spatial correlation and encased in poorly ordered matrix. Bone deposited on top of this has lamellar collagen orientation as well as a layered arrangement of osteocytes, both parallel to the surfaces of the initial tissue. This supports the hypothesis that the initial bone constitutes an endogenous scaffold for the subsequent deposition of parallel fibered and lamellar bone.     

Electrical Signal Transmission in a Bone Cell Network: The Influence of a Discrete Gap Junction

A refined electrical cable model is formulated to investigate the role of a discrete gap junction in the intracellular transmission of electrical signals in an electrically coupled system of osteocytes and osteoblasts in an osteon. The model also examines the influence of the ratio q between the membrane's electrical time constant and the characteristic time of pore fluid pressure, the circular, cylindrical geometry of the osteon, and key simplifying assumptions in our earlier continuous cable model (see Zhang, D., S. C. Cowin, and S. Weinbaum. Electrical signal transmission and gap junction regulation in a bone cell network: A cable model for an osteon. Ann. Biomed. Eng. 25:379–396, 1997). Using this refined model, it is shown that (1) the intracellular potential amplitude at the osteoblastic end of the osteonal cable retains the character of a combination of a low-pass and a high-pass filter as the corner frequency varies in the physiological range; (2) the presence of a discrete gap junction near a resting osteoblast can lead to significant modulation of the intracellular potential and current in the osteoblast for measured values of the gap junction coupling strength; and (3) the circular, cylindrical geometry of the osteon is well simulated by the beam analogy used in Zhang et al. © 1998 Biomedical Engineering Society. PAC98: 8722-q, 8710+e