If you were to slice open a dozen bones of various sorts and wash away the marrow with a high-pressure stream of water, it would be clear that the remaining bone is of two distinct kinds, as determined by porosity (the volume fraction of soft tissues). While in principle the porosity of bone can vary continuously from 0 to 100 %, in fact most bone tissues are of either very low or very high porosity, with little bone of intermediate porosity. These two types of bone tissue are referred to as compact bone and cancellous bone, respectively (Fig. 2.3a).

Cancellous bone (also called trabecular or spongy bone) is porous bone found inside cuboidal bones (e.g. vertebrae, carpal bones), the flat bones (e.g. ilium, sternum), and the ends of long bones (Fig. 2.3b); its porosity is 75–95 %. The pores are interconnected and filled with marrow. The bone matrix is in the form of plates or rods called trabeculae, each about 100–150 μm thick. The arrangement of the trabeculae is variable. Sometimes they appear to be organized into orthogonal arrays; often, they are more randomly arranged. Cancellous bone tissue has a much lower density than cortical bone and allows the skeleton to build large but lightweight conical volumes of bone at the ends of long bones that carry the large loads of the joints with low stress at the wide end (the metaphysis) and higher stresses in the narrower shaft (the diaphysis). The density of the bone increases as the cone narrows, creating a means for the bone to funnel the high loads imposed at the joint to the stronger and more massive cortical bone comprising the middle portion of the bone (i.e. the diaphysis).

Compact bone is the dense bone found in shafts of long bones and forming a cortex or shell around vertebral bodies and other cuboidal and flat bones (Fig. 2.3a, b). Hence, it is also called cortical bone. It is strong and provides both support and protection. Its porosity is 5–10 %, although this increases with age and with osteoporotic changes to the skeleton. Its pores consist of spaces categorized as follows.

Haversian canals are approximately aligned to the long axis of the bone, contain capillaries and nerve fibers, and are about 50 μm in diameter (about the diameter of a human hair). Haversian canals are named after an English physician, Clopton Havers (1691).

Volkmann’s canals are short, transverse canals connecting Haversian canals to each other and to the outside surfaces of the bone. These canals also contain blood vessels and nerve fibers. They are named after Richard von Volkmann (1830–1889), a surgeon and early advocate of Lister’s antiseptic surgical methods.

Resorption cavities are the temporary spaces created by osteoclasts in the initial stage of remodeling (see Chap. 3). Resorption cavities are about 200 μm in diameter.

It is important to remember that bone is a dynamic porous structure; its porosity may change as the result of a pathologic condition, with age, or in a normal adaptive response to a mechanical or physiologic stimulus. Cancellous bone may become more compact, or compact bone may become more porous. Such changes strongly affect bone’s mechanical properties.

Examining compact and cancellous bone at a still finer scale of resolution, it is evident that each may contain two major types of bone tissue.

Lamellar bone is slowly formed, highly organized bone consisting of parallel layers or lamellae comprising an anisotropic matrix of collagen fibers interdigitated with mineral plates. There may be a variety of patterns of lamellar arrangement at the level of the collagen fibril, and these likely each have important mechanical implications (Reisinger et al. 2011). Microscopically, however, the lamellae create optically bright circumferential bands of bone, each 3–7 μm thick, that give the appearance of tree rings, each separated by an optically darker layer about 1 μm thick that is mechanically less stiff than the thicker lamellae (Tai et al. 2007). The lamellae may be arranged around the endocortical or the periosteal circumference of the bone (primary or circumferential lamellae), or may be arranged concentrically around individual vascular channels (osteonal or concentric lamellae).

Two fundamentally different kinds of “plywood” architecture coexist within the general complexity of bone’s lamellar structure (Giraud-Guille 1988). The first of these schemes corresponds to the classical view of lamellar structure: the collagen fibers are parallel in each lamella and change direction by 90° at the lamellar interface. That is, as a lamella is built up, layers of collagen are put down in one orientation. Then, when the next lamella is started, the orientation of collagen fibers suddenly changes so that they are laid down at right angles to the previous direction. In other regions of the bone, this orthogonal plywood-like structure is replaced by helicoidal plies (Fig. 2.4). In this scheme the collagen fibers continuously change their direction, so that in a sense there are no individual lamellae. However, the bone still shows a lamellar structure because as the orientation of the collagen fibers rotates through 180° cycles, the fiber orientation repeats itself, and layers appear when a histologic section is examined microscopically. A helicoidal arrangement of collagen fibers leads to a transversely isotropic structure at this level of organization as the elastic properties are similar in different directions (Reisinger et al. 2011). In her landmark paper, Giraud-Guille (1988) showed that both these architectures are present in human compact bone, but their relative distribution and interspecies variation have not been determined.

Both these forms of lamellar structure give rise to birefringence, the capacity of some fibrous structures to interact with polarized light. When a histologic section of bone is transilluminated with polarized light and viewed through a polarizing filter oriented perpendicular to the vibration plane of this incident light, the section appears dark (i.e., the observer sees a “dark field”) except where collagen fibers are parallel to the plane of the section. These collagen fibers rotate the light’s plane of polarization so it is no longer perpendicular to the viewing polarizing filter. Therefore, the light is not blocked and reaches the viewer’s eyes. Thus, in a bone section observed in a polarizing microscope, transversely oriented fibers are bright and longitudinally oriented fibers are dark. In the 1960s, Ascenzi and his co-workers used this phenomenon to categorize osteons as having bright, alternating, or dark lamellae (corresponding to their collagen fibers exhibiting mostly circumferential, alternating, or longitudinal orientation) (Ascenzi and Bonucci 1967) . These classifications are shown as parts a, b, and c, respectively, in Fig. 2.5. A dark “iron cross” pattern superimposed on the brighter osteons shows where the lamellae lie parallel to the polarization directions of the upper or lower polarizing filters.

Woven bone is a quickly formed, poorly organized tissue in which collagen fibers and mineral crystals are more or less randomly arranged. Woven bone may become more highly mineralized than lamellar bone, which, mechanically speaking may help compensate for its lack of organization. Woven bone is common in a fracture callus and is formed early in the healing process to help stabilize the broken bone. In this case, woven bone can be considered calcified scar tissue that is rapidly formed for damage control and then remodeled and replaced over time with lamellar tissue reflective of the site’s original organizational structure. The important generalization is that woven bone can be made more quickly than lamellar bone, but is weaker. Rats and other small animals also have fine-fibered bone in the cortices of their bones. In this bone the rather randomly arranged collagen fibers are smaller and closer together than in woven bone, so the tissue appears to be more organized (Fig. 2.6). It may be that the lamellar organization of bone can vary continuously; depending on factors such as how fast it is made.

Compact bone may be further characterized as primary or secondary bone.

Primary bone is tissue laid down de novo on an existing bone surface, such as the periosteal surface, during growth. It may be of two general types:

Circumferential lamellar bone, in which the lamellae are parallel to the bone surface (Figs. 2.7a–c). Blood vessels are incorporated into the lamellar structure such that each is surrounded by several circular lamellae, forming a primary osteon with a primary Haversian canal at its center.

Plexiform bone occurs when the rate of formation is greatly increased by continually constructing a trabecular network on the surface and filling in the gaps. The result is a mixture of woven bone (the trabeculae) and lamellar bone (the filled-in spaces). Plexiform bone contains rectilinear residual vascular spaces, which often produce a “brick wall” appearance. Figure 2.8 contains brightfield and polarized light photomicrographs of this structure. It typically occurs in large, fast-growing animals like cows. Racehorses, which put enormous stress on some of their bones, have a similar kind of bone that may have exceptional fatigue resistance (Stover et al. 1992).

Secondary bone results from the resorption of existing bone and its more or less immediate replacement by new, lamellar bone. This process is known as remodeling (see Chap. 3). In compact bone, secondary tissue consists of cylindrical structures known as secondary osteons or Haversian systems (Fig. 2.9a). These are about 200 μm in diameter, although this varies with age in predictable ways (Currey 1964; Jowsey 1966; Martin et al. 1980), and consist of about 16–20 cylindrical concentric lamellae surrounding a central Haversian canal. Osteons are about 1–10 mm long and in the human femur they run at an angle of 11–17° with respect to the long axis of the bone (Cohen and Harris 1958). The boundary between the osteon and the surrounding bone is known as the cement line. It is composed largely of glycosaminoglycans and can be either poorly mineralized (Schaffler et al. 1987) or highly mineralized (Skedros et al. 2005). The cement line can act as a weak boundary that traps cracks to slow crack growth in cortical tissue (Burr et al. 1988). The weak interface between the osteon and the rest of the bone matrix increases tissue toughness and fatigue life. A cement line with low mineralization can be a viscous material that may relieve locally high shear stresses. The low shear stiffness reduces the ability of a cement line to transmit energy to a growing crack. Displacement (creep) of cement lines has been observed in human bone over long periods of torsional loading (Lakes and Saha 1979). In adult humans, most compact bone is entirely composed of secondary bone, which may include whole osteons and the remnants of older osteons that have been partially resorbed (interstitial bone). Remodeling of individual trabeculae within cancellous bone, which occurs frequently in both children and adults, rarely produces a complete osteon because the 200 μm diameter osteon is too large to fit within most trabecula of average diameter 100–150 μm. Instead, hemi-osteons are formed (Fig. 2.9b). One surface of the hemi-osteon borders the marrow cavity, rather than a Haversian canal, but the remaining surface is separated from older bone within the trabecula by a cement line just as whole osteons are separated from their surroundings in compact bone.

We have seen that bone contains three primary constituents: mineral, an organic matrix that is largely collagen, and water. Clearly, these three substances have distinctive physical properties that contribute to the overall mechanical behavior of the tissues they comprise. Architectural organization and substrate interactions are also important contributors to skeletal performance but the relative masses of bone’s three primary constituents are a good indicator of bone health and enable a reasonable first approximation of its intrinsic strength and stiffness.

Simple gravimetric analysis is a time-honored method of determining the mineral, organic and water content of extracted bones. In this method bone specimens are precisely weighed before and after drying in an oven at 100 °C to determine wet weight, dry weight and percent water. The specimen is then placed in a muffle furnace and heated to over 600 °C which volatizes all organic constituents and removes any remaining bound water leaving the ash mass which is weighed to determine the mineral component of the tissue. The organic content (predominately collagen) and ash content (mineral) are then determined as a percentage of the original wet weight. Tissue mineralization or ash fraction is calculated as the percentage of ash weight to dry weight. The ash fraction is independent of bone porosity and is an excellent measure of tissue mineralization, which varies by species, skeletal site, age and disease. Collagen content can be measured as the difference between total dry weight and ash weight, divided by dry weight. The gravimetric method can be extended to measure carbonate content by heating specimens to above 900 °C, the temperature at which incorporated carbonate decomposes to CO₂.

Gravimetric analysis yields good quantitative information about the major constituents of bone but it cannot measure the quality of collagen or mineral in the sample, nor can it provide architectural information related to bone density and porosity. The average weight density of the mineralized matrix (ρbone) can be determined in the laboratory using Archimedes law of buoyancy using a fluid of known weight density (ρfluid = 1 g/cm³ for distilled water; Eq. 2.1) provided that all marrow elements and fat are removed beforehand.
The density of either cortical or trabecular bone specimens can be measured using this approach, but completely removing marrow elements and fat from highly porous trabecular samples is difficult. Apparent density, the weight of the sample divided by its total overall volume (typically a machined cube or cylinder), including the pores and spaces within, is a far more common measure for trabecular samples. Once the apparent density is known the porosity (P) of the sample can be determined using the following equation:
where ρapparent is the apparent density of the sample and ρbone is the density of the bone tissue in the sample as determined directly using Archimedes principle or another technique. A generally accepted value of 2.0 g/ml is frequently substituted for direct measurements of ρbone, thereby allowing for easily obtained estimates of porosity.

The porosity (P) of a bone sample is simply the percentage of total sample volume not occupied by bone tissue. Conversely, the bone volume fraction (BV/TV) of a sample is the percentage of volume occupied by bone matrix. The material comprising the bone volume fraction is often referred to as bone tissue to distinguish it from the term “bone,” which usually refers to a region large enough to contain Haversian canals, marrow spaces, or other voids. Cancellous bone has been somewhat arbitrarily defined as having porosity greater than 0.5, with free space largely occupied by marrow elements; in cortical bone, porosity is less than 0.5 and space is defined by Haversian and Volkmann’s canals and resorption cavities created by osteoclasts. For practical reasons, the smallest voids in bone, consisting of octeocyte lacunae and canaliculi, have traditionally been included in the bone volume fraction rather than the porosity fraction.

The foregoing describes weight-based measures of bone volume, density and porosity. Another traditional method of analysis employs histologic sections and the principles of stereology. Before the advent of automated procedures this approach typically entailed overlaying a two-dimensional histologic image of a bone section with a series of lines or an array of points and measuring the length of line passing through, or the number of points landing on, bone or void space. When enough sections are sampled the approach yields accurate estimates of bone composition because the ratio of bone tissue to void space remains the same whether assessed by counting points (0D), measuring line lengths (1D), measuring areas (2D) or measuring volumes (3D). Bone volume, bone density and a host of other architectural measures such as bone surface area, trabecular number, thickness and trabecular spacing are now easily made using newer 3D imaging modalities and automated procedures.

A bone can change shape both from the outside and the inside as a result of age, diet, disease, fracture and from many other causes. Today, there are many ways to image the shape of a bone in a person or animal without harming them. X-Ray Computed Tomography (CT or “Cat Scan”) is often the best method for seeing bones inside the body. The machines used in hospitals ordinarily have the resolution to image clearly objects down to about a millimeter, which is outstanding for many medical purposes but insufficient to see trabeculae, osteons, Volkmann’s canals (blood vessels) and other microanatomy in bones that contribute to its health and strength.

There now are many commercially available CT scanners for small specimens that have better than one millimeter resolution. The highest resolution machines are often called “X-Ray Microcomputed Tomography” scanners or “MicroCT” scanners for short. For resolution on the order of 35–50 μm there are machines that can be used to image the bones of small animals such as mice or rats while they are alive. For finer resolutions on the order of 1–10 μm the required x-ray doses are dangerous to life making high resolution scanning usually restricted to dead or nonliving specimens.

The three dimensional (3D) images obtained from microCT (Fig. 2.11) are used to understand bone microarchitecture in many ways. One method is to use computer assisted image processing to measure bone density or connectivity and to count trabeculae or other features directly from the image (Hildebrand et al. 1999). In the past, measurement of the interior of a bone specimen was done by physically sectioning the bone, mounting the thin histologic bone specimens onto microscope slides and using stereometric analyses (Parfitt et al. 1987; Russ and DeHoff 2000) to estimate 3D information from the 2D microscopic images. MicroCT scanning and image analysis has eliminated much of the need to use bone histology for measuring the interior microanatomy of bone, although histology is still necessary to image bone cells since microCT cannot resolve them adequately. Histology is, of course, still necessary to measure cell-level information such as gene expression and protein distribution, or to quantify dynamic bone remodeling.

A powerful tool for understanding how bone microstructure contributes to tissue strength and stiffness is to convert the 3D microCT image into a finite element stress analysis model (Fig. 2.12). Finite element models of bone tissue built in this way are much better at predicting the mechanical properties of bone tissue than are simpler models that don’t include microanatomy (Hou et al. 1998; Homminga et al. 2002; Zauel et al. 2006). Prior to the invention of high resolution microCT finite element modeling of trabecular bone in 3D was extraordinarily time consuming or impossible. Now, however, stress analysis is a normal part of research into what makes bones weak or strong. With continued technological development, higher resolution CT scanners may become available to hospital patients and some of the insights from microCT could become useful to predicting the risk of bone fractures for individual patients.