Histology, Bone

Structure

As a specialized connective tissue, bone is comprised of mainly type 1 collagen fibers and inorganic salts. Type 1 collagen is a non-calcified matrix designed to resist the tension experienced by the weight-bearing functions of bone. It forms from osteoblasts, the main cellular component of bone. 

Osteoblasts function in the production, transport, and arrangement of the osteoid matrix. Furthermore, they initiate and regulate matrix mineralization and control the neighboring bone cells activities.  Osteoblasts morphology is closely related to their metabolic status. They are spindle-shaped when quiescent. When active, they are polyhedral, with abundant basophilic cytoplasm intimately in contact with the bone. They have a conspicuous nucleolus and a prominent perinuclear halo (developed Golgi apparatus).[2][3][4]

Once the osteoid is mineralized, the precursor cells get surrounded by organic intracellular substances called lacunae to become fully developed and matured into osteocytes. The mineral content is chiefly hydroxyapatite crystals composed of mainly calcium and phosphate ions but can occasionally have sodium and carbonite as well.[5][6]

Osteocytes have a widespread distribution. Their cell processes are fundamental to allows osseous tissue to be responsive to the mechanical and metabolic organism constraints. Histologically, they have long and delicate cytoplasmic processes and small nuclei, not always visible in every plane of section. Their number, size, shape, and position vary according to bone type. In fact, in woven bone, they are numerous, large, and disorganized, whereas, in lamellar bone, they are fewer in number, smaller, spindle-shaped and architecturally regular.[7][8]

The osteon, anatomically, is represented as the single functional unit of bone tissue. It is arranged with concentric lamellae of collagen fiber orientations around a central canal consisting of osteocyte’s arterial, venous, and nerve supply, is known as the Haversian canal. This system also consists of the canaliculi and Volkmann canal which allow for communication between neighboring osteocytes and communication between neighboring osteons, respectively.

With a mechanism responsible for building up bone, osteoclasts are the large, multinucleated cells on the surface of bones in pits called Howship lacunae responsible for removing calcium from bone by secreting proteolytic enzymes and hydrogen ions to dissolve the calcium hydroxyapatite crystals.[5] 

On average, osteoclasts have 12 nuclei, though the number may range from 2 to as many as 100. In their cytoplasm, interconnecting actin filaments produce a clear area between the cell membrane and the nuclei.[9]

Architecturally, bone categorizes into two subtypes: cortical and trabecular bones. Similarly, the primary mechanical determinants of the strength of bone are specific for the subtype as described: width and porosity for cortical bone, and shape, width, connectivity, and anisotropy for trabecular bone.[5] Further differentiations regarding the two subtypes are as follows[6][10]:

1. Cortical Bone: consists of about 80% of the total bone in the body and is much stronger than trabecular bone. It is very resistant to bending, torsion, and compression and is much more dense with a minimal role in metabolism. It is seen mostly in the shaft of long bones like the femur and the tibia as well as in the outer shell of trabecular bone.
2. Trabecular Bone: consists of only 20% of the total bone but has ten times the surface/volume ratio of cortical bone. It responds eight times faster to changes in load making it far more dynamic. It occurs in areas that more subject to compression such as the vertebral body, pelvis, and the metaphyses.

Function

There are three main functions of the human skeletal system classified into the mechanical, formation of hematopoietic cells, and metabolism.[4]

1. Mechanical: Bones provide a frame for other soft tissues of the musculoskeletal system to attach to such as muscles, tendons, and ligaments. These allow for support as well as the movement by contracting and relaxing of the muscles which then, in turn, result in flexion, extension, abduction, adduction, and other forms of movement. They also help form a mechanical barrier to different structures within the human body. For example, the rib cage and the skull help shield our vital organs, the heart/lungs, and the brain, respectively, from trauma.
2. Formation of hematopoietic cells: The marrow is found in the trabecular portions of bones and is responsible for hematopoiesis, or the production of red blood cells, white blood cells, and platelets.
3. Metabolism: The bone matrix can store several minerals, chiefly calcium and phosphorus as well as iron in the form of ferritin. Chondroitin sulfate, a carbohydrate moiety, is also a commonly found element in the matrices. Specific growth factors, including insulin-like growth factor or IGF-1, are housed in bone and then released periodically. pH balance is also regulated as bones may alter the composition of alkaline salts in the serum to maintain the optimal pH level. Moreover, osteocytes can engulf toxic molecules and heavy metals from the serum as a means of detoxification.[11]

Tissue Preparation

The best way to thoroughly observe the microarchitecture of bone is to perform undecalcified bone histology, which can reveal the mineralized and cellular components of the bone as well as patterns regarding the physiological processes of bone formation and resorption. This process, in turn, allows for histological techniques such as fluorochrome analysis and histomorphometry to further probe and observe the specimens.

Performing undecalcified bone histology varies from the techniques utilized in conventional paraffin embedded methods; this is because of density and lower permeability of the tissue, demanding more time-consuming fixation and processing times. The tissue is first placed in 10% phosphate-buffered formalin solution. Using a band saw, the specimen is cut to size and then placed in an opaque container, submerged in ascending concentrations of ethanol from 70 to 100% for one week at a time, increasing in ten percent increments. Afterward, the specimen is cleared in butanol for one week. It is imperative that the densities of the tissue and resin be similar., which can be done by using a test specimen beforehand and then modifying it the resin density afterward. Generally, a 2-hydroxyethyl methacrylate based preparation solution is the choice when assessing through light microscopy while methyl methacrylate is for immunohistochemistry.

Ground sectioning is prone to yield larger sections, ranging from 20 to 50 microns in thickness which makes it advantageous if intending to use fluorochrome analysis. The sledge microtome, on the other hand, tends to produce thin sections, more ideal for light microscopy.[12]

Histochemistry and Cytochemistry

Beyond the more widely used hematoxylin and eosin histochemical staining, several other stains are more advantageous in certain situations that warrant bone biopsies. This includes[13][14][15][16][17][16][15][14][13]:

• Alkaline phosphatase: useful in understanding relative osteoblast activity
• Von Kossa: best utilized to observe the degree of mineralization in tissue samples and cell cultures
• Toluidine blue O: used to identify mineralized bone and osteoid seams, osteoblasts, and osteoclasts
• Safranin O: stains proteoglycan content in osseous tissue samples
• Tartrate-resistant acid phosphatase (TRAP): enzyme specific stain for the presence of osteoclasts
• Prussian blue: stains for iron content in bone tissue specimens
• Elastin/Van Gieson (EVG): stain used to determine the presence of elastic fibers
• Calcein: labels bone mineralization fronts in vivo
• Oil red O: stain used to visualize the presence of adipose cells in bone specimens

Microscopy, Electron

Through recent uses of transmission electron microscopy, ion-milled samples of cortical bone have demonstrated polycrystalline mineral structures enshrouding the collagen fibrils. Approximately 80% of the minerals, composed of hydroxyapatite juxtaposed with type 1 collagen, are outside the fibrils. The remaining amount of minerals that resides inside the fibrils are shown to be in the gap zones. These gap zones are between the C and N terminals of the collinear collagen proteins in the fibrils. Their arrangement is as crystals that are not as large as those outside the fibrils.[18]

Electron tomography is also an alternative that utilizes bright-field images to build 3D models. In tissue samples where the mass thickness is dominant, such as in bone, the lattice planes from the crystals produce Bragg diffractions that lead to visualizations of wavering thicknesses. High-angle annular dark-field (HAADF) imaging, or Z-contrast imaging, is, likewise, another way of conceptualizing tomographic images of osseous tissue and is very useful in understanding the 3D relationship amongst the different biochemical systems at play.[19]

Clinical Significance

Bone tissue is susceptible to a myriad of pathologies that may range from etiologies of embryological, metabolic, autoimmune, neoplastic, or idiopathic origins. These include, but are not limited to, the conditions discussed below.

Achondroplasia is a genetic disorder commonly associated as a cause of dwarfism. Individuals affected may present with short extremities due to decreased development of endochondral bone; this is the type of bone that is responsible for the growth of long bones in the body. This formation begins with the construction of cartilage matrices which are eventually supplanted by new forming bone. However, clinically, these patients present with normal-sized head and chests. This presentation is because the development of intramembranous bone is unaffected. These bone processes are responsible for forming flat bones – skull, sternum, and rib cage. They do not require a preexisting cartilage matrix to lay down new bone tissue in its place. Research has identified the cause as an activating mutation in fibroblast growth factor receptor 3, or FGFR3, gene. This gene is, under normal circumstances, inhibited to allow for standard bone growth. However, with a lack of inhibition of this gene, bone growth is arrested leading to the phenotype.[20]

Paget disease of the bone is characterized to be an imbalance amongst the activities of osteoblasts and osteoclasts. Of unknown etiology, the condition only affects localized portions of the skeletal tissue, generally involving one or more neighboring bones rather than the diffuse skeletal system. The disease process has three different stages. Osteoclast activity first increases, leading to a higher breakdown of bone tissue than normal. From this, the negative feedback mechanism is activated leading to osteoblasts responding to the breakdown and beginning to lay down bone. Eventually, osteoblastic activity is uncontrolled and continues to lay down bone tissue far past the metabolic requirement yielding a thick, sclerotic bone that is susceptible to fractures. Clinical symptoms are a result of the end process of this overgrowth of bone such as bone pain from microfractures, hearing loss due to impingement on the vestibulocochlear nerve from excess bone, and lion-like facies following the involvement of the craniofacial bones. Alkaline phosphatase becomes elevated more than twice the normal level juxtaposed with the clinical features, in a patient should lead a physician to consider Paget as a diagnosis because an isolated elevation of alkaline phosphatase in adults above 40 years most commonly points to the disease. Bone specimens of these patients illustrate a mosaic pattern of lamellar bone representing the low threshold required to fracture.[21]

If untreated, Paget disease of the bone can act as a risk factor for osteosarcoma, a malignant proliferation of osteoblasts. Much more common in teenagers than in the elderly, this skeletal neoplasm begins in the metaphysis of long bones with patients complaining of bone pain with swelling or as a pathologic fracture (a break in the bone caused by weakness of the bone through disease rather than trauma). According to the literature, the most common bones affected are the distal femur and the proximal tibia composing of bones of the knee joint. Radiological investigations of osteosarcomas depict a “sunburst” appearance of a destructive mass in affected areas. Due to the over-proliferation of osteoblasts, the periosteum is shown to be lifted, forming a Codman triangle. Levels of serum alkaline phosphatase and lactate dehydrogenase are also elevated in these patients as they are indicative of increased osteoblast activity. The degree of the elevation in these markers also determines the prognosis for patients, as higher levels signal more advanced disease. A biopsy is necessary for these patients. This biopsy can be either a closed biopsy through fine needle aspiration cytology (FNAC), a core needle biopsy, or an open/incisional biopsy which would allow an appropriate amount of tissue extraction for subsequent ancillary studies. Histological imaging demonstrates pleomorphic cells with the ability to produce osteoid, confirming the diagnosis.[22]