Histology of Bone
The strength, shape and stability of the human body are dependent on the musculoskeletal system. The most robust aspect of this unit is the underlying bony architecture. Bone is a modified form of connective tissue which is made of extracellular matrix, cells and fibers. The high concentration of calcium and phosphate based minerals throughout the connective tissue is responsible for its hard calcified nature. The histological structure, mode of ossification, cross-sectional appearance, and degree of maturity influences the classification of bony tissue.
In addition to discussing the cellular constituents of bone and the architectural arrangement of their products, this article will also address the embryology and mechanisms of ossification as well. Furthermore, some prominent bone-related pathological processes will also be addressed.
Please take into account that unlike most organ systems that complete organogenesis during the antenatal period, skeletal development is spread out over the gestational period and continues into extra-uterine life. Bone is derived from three embryonic sources. The neurocranium and the viscerocranium originate from derivatives of the neural crest cells as well as paraxial mesoderm. The paraxial mesoderm also contributes to the formation of the axial skeleton, while the appendicular skeleton originates from the lateral plate mesoderm.
The so-called flat bones of the body such as calvaria, mandible, maxilla, etc. and long bones such as those of the limbs, are formed by two different processes. The former originates by way of intramembranous ossification, while the latter undergoes endochondral ossification. The initiation of either process depends on the differentiation of the preceding mesenchymal cell line. If the mesenchymal cells differentiate into chondrocytes, then endochondral ossification will occur. However, should the mesenchymal cells differentiate into osteoblasts intramembranous ossification would ensue.
Irrespective of the pathway taken, ossification begins around the 6th or 7th gestational week and persists well into extra-uterine life; the clavicle can take up to 20 – 21 years for complete fusion to occur, and 26 years for the epiphyseal scar to disappear. Extra-uterine bone development has been classified into 5 stages.
- In stage 1, the epiphysis is not yet ossified.
- Once ossification becomes apparent in the epiphyses, then the bone is in stage 2 of development.
- At the point where the epiphyses and diaphysis begin to fuse, then the bone has entered stage 3.
- Stage 4 represents complete fusion of the epiphyses and diaphysis, leaving behind an epiphyseal scar at the site of the epiphyseal growth plate.
- The final stage is characterized by disappearance of the epiphyseal scar.
Endochondral ossification relies on an analgen in the form of hyaline cartilage laid down during embryogenesis. Initially, a hyaline cartilaginous framework is laid down as a template for osteogenesis. It is encased by a perichondrial layer that comprises of a condensed vascular mesenchyme. The model grows by both interstitial (replication of chondrocytes and secretion of new matrix) and appositional (absorption of old cartilage and deposition of new matrix) methods. The chondrocytes (cartilaginous cells) in the mid shaft of the cartilaginous template (diaphysis) begin to replicate and hypertrophy. An increase in the number of vacuoles can be observed in the cytoplasm in this phase. Subsequently, the matrix is compressed, forming thin fenestrated septae. The cartilage model subsequently calcifies, resulting in decreased diffusion of nutrients to the cells. They eventually degenerate, die, and calcify; leaving confluent lacunae in their absence.
As the cartilage calcifies, the inner layer of the perichondrium begin to express osteogenic (i.e. bone forming) properties; and thus become osteoblasts. Osteoblasts are responsible for production of bone matrix; they eventually produce a bony collar around the diaphysis called the periosteal collar. The connective tissue superficial to the periosteal collar is subsequently referred to as the periosteum. The visceral periosteum contains mesenchyme cells that evolve into osteoprogenitor cells. This cell line replicates and further differentiates into osteoblasts. These cells travel with osteogenic buds, which are terminal capillary sprouts.
The osteoclasts break down previously formed bone and as a result, facilitate the breakdown of calcified cartilage to allow invasion of osteoblasts (that will lay down new bone matrix) and osteogenic buds (to establish nutrient supply to the developing bone). The inner surface of bone (i.e. endosteum) that lines all bony cavitation is also covered by a single layer of osteoprogenitor cells that provides a supply of stem cells for future differentiation.
The bony development occurring in the diaphysis is referred to as the primary ossification centre. However, as the bones continue to elongate and increase in diameter, a secondary ossification centre develops in the epiphyses (distal articular ends) of the long bones. The secondary ossification centre is also invaded by a vascular supply and mesenchymal derivatives similar to those present in the primary ossification centre.
At both the primary and secondary ossification centres, cartilage is replaced by bone. However, there is a region where cartilage is preserved, known as the epiphyseal growth plate. The bone continues to grow by appositional and interstitial mechanisms at this region until the ideal length is achieved. Fusion of the epiphyses with the diaphysis marks the cessation of bone growth. At this point, the only remnant of hyaline cartilage is found at the articulating surfaces of the bone.
Unlike endochondral ossification, the intramembranous ossification pathway does not require a cartilaginous scaffold. Instead, the bone is formed within primitive mesenchymal layers that have rich blood supplies. The stem cells within the mesenchyme differentiate into osteoprogenitor cells that replicate adjacent to capillary beds. The end result is scattered layers of osteoblasts producing bone matrix. Consequently, there are multiple ossification centres observed in intramembranous ossification. The osteoblasts are described as being polarized cells, owing to the fact that osteoid secretion occurs at the surface furthest away from the blood supply. The ossification centres subsequently anastomose, leaving a woven pattern of trabeculae, referred to as primary spongiosa or spongy bone.
Functional Bone Constituents
There are two regions within bone that contains osteoprogenitor cells and their derivatives, along with osteoclasts and other cells involved in bone homeostasis. These are the periosteum and the endosteum. The periosteum is a fibro-collagenous layer at the outermost layer of the bone. It is anchored by Sharpey’s fibres (collagen fibres) and found along the outer surface with the exception of the articular surfaces of the bone and areas of ligament and tendon insertion. About two to three layers of osteoblasts occupy the space between the visceral periosteum and the newly produced bone matrix. The periosteum is actively involved in the repair of fractures; in areas where it is absent (intracapsular areas) the fractured bones heal at a slower rate.
The periosteum actively participates in bone development in utero. However, it is the endosteum that produces more osteoprogenitor cells and osteoclasts that facilitate bone remodelling. The osteoblasts at the endosteum are flat and are surrounded by type III collagen. It extends along the inner surface of the bone; projecting even into the Haversian canals.
Osteoclasts are thought to be monocyte derivatives that have the responsibility of removing bone during growth and remodelling. They are larger than osteoblasts and osteocytes, polymorphic and multinucleated (with roughly 20 oval nuclei in the cytoplasm). They are commonly found in Howship’s lacunae (resorption bays). Owing to the high metabolic demand of these cells, there are numerous mitochondria in the cytoplasm. Additionally, there are many vacuoles that contain acid-phosphatase enzymes that facilitate bone resorption.
There are many microtubular structures that facilitate the transportation of lysosomes to the Golgi body and deeper ruffled membrane of the osteoclast. The ruffled membrane is the site of osteoclast activity, where hydrogen ions are released along with collagenase (non-lysosomal enzyme) and cathepsin K (lysosomal enzyme), resulting in breakdown of bony material. These cells are activated by osteoblast signals (discussed below), calcitriol and parathyroid hormone levels, and are inhibited by calcitonin from the thyroid C cells.
Osteoblasts are mesenchymal derivatives that are differentials of osteoprogenitor cells. The latter are stimulated by bone morphogenic proteins just before bone begins to form. Unlike the osteoclasts, osteoblasts are mononuclear, cuboidal and basophilic cells that are found on the developing surface of bone during growth or remodelling. Osteoblasts secrete and also facilitate the mineralization of osteoid matrix.
Because of the need for newly formed osteoblasts to move to areas of bone growth and remodelling, the cytoplasm is filled with actin and myosin bundles. There are dendritic extensions from the cytoplasm that communicate with neighbouring osteoblasts, thus establishing electrical and metabolic continuity among the osteoblasts and osteocytes within a system. It should be noted that osteoblasts express receptors for calcitriol and parathyroid hormone. Activation of the parathyroid hormone receptors result in osteoblast-induced differentiation of immature osteoclasts.
Osteoblasts become trapped in the bone matrix that they produce. Subsequently, they differentiate into osteocytes. These cells retain the cytoplasmic projections and form numerous communications with neighbouring osteocytes and osteoblasts. Unlike chondrocytes, osteocytes neither undergo cellular division, nor produce new matrix. These cells are elliptical, mildly basophilic and contain an oval nucleus with fewer organelles than osteoblasts.
The structural layout of bone can be classified in one of the following groups: either trabecular (cancellous or spongy) or compact. Histologically, spongy bone is comprised of anastomosing strips of slender bone known as trabeculae that enclose marrow and blood vessels. It forms the relatively softer core of the bones that is filled with marrow. The less densely arranged trabeculae also contribute to making the bones lighter (as opposed to the heavier compact bone). Communication between adjacent cavities is achieved by canaliculi. Although the trabecular network makes the bone lighter, and increases the available space to house marrow, the arrangement also provides reinforcement for the bone, making it stronger.
Compact bone stands in stark contrast to trabecular bone in several ways. The functional units of compact bone are osteons; which contain a centrally located Haversian canal, encased in lamellae (concentric rings). Osteocytes can be observed in the lacunae between the osteons. The osteons – unlike the trabeculae – are densely packed, making compact bone tougher and heavier than spongy bone. The Haversian canals facilitate passage of blood vessels supplying the developing bone.
Bone remodelling is an ongoing process that occurs throughout life and is dependent on the relationship between bone deposition and resorption; both of which are linked to the activity of osteoblasts and osteoclasts. After groups of osteoclasts dissolve an area of bone, the osteoblast will begin to deposit bone matrix in that area, ensuring that a blood vessel is within the region of deposition.
There is a variety of bone disorders that have deleterious effects on patients. Some common ones will be discussed below:
Osteopenia is a disorder of bone mass characterized by bone density that is 1 to 2.5 standard deviations below the age appropriate average for that individual. Once the bone density is more than 2.5 standard deviations below the average, then the patient is said to have osteoporosis. However, the presence of non-traumatic fractures is highly indicative of osteoporosis. Bone density usually peaks in young adulthood and gradually declines as the patient ages. Therefore, age-related or senile osteoporosis is not uncommon. Postmenopausal women are also at increased risk of osteopenia and osteoporosis owing to the oestrogen deficiency. Other causes include poor calcium intake and physical inertia.
Paget’s disease of the bone stands in contrast to osteoporosis in that it is a disorder of increased, disorganized bone production. The sequel follows three steps, commencing with an osteolytic phase initially, then a subsequent osteoclastic-osteoblastic picture that culminates with an osteoblastic overtone. Finally, the disease usually ends with a osteoclastic phase. While the aetiology is unknown, there is evidence of both genetic and environmental contributors to its pathogenesis.
Lack of vitamin D
Vitamin D plays an important role in calcium metabolism. Lack of vitamin D therefore results in two age-dependent disorders. In children with vitamin D deficiency, there is an abnormal mineralization of bone matrix at the epiphyseal plate. The resultant disorder is known as Rickets. When this disorder occurs in adults, the matrix deposited during bone remodelling is inadequately mineralized. This disorder is known as osteomalacia.
Osteomyelitis is an infectious disorder of the bone, resulting in an inflammatory response. While it can occur as a result of a local insult (i.e. posttraumatic infection of an open fracture), haematogenous spread to the bone following a systemic infection also occurs. Special care should be taken when managing immunosuppressed patients (diabetics, iatrogenic immunosuppression, HIV, etc.) with fractures or systemic infections.
The parathyroid gland releases parathyroid hormone in order to establish calcium homeostasis. This is implemented via several mechanisms, including:
Upregulation of RANKL in osteoblasts results in stimulation of the RANKL receptors on the primitive osteoclasts. This results in activation of osteoclastic activity and subsequent bone resorption.
More calcium is reabsorbed by the renal tubules
Stimulation of vitamin D synthesis by the kidneys
Promotes excretion of phosphate ions by the kidneys (recall phosphate ions impair alpha-1-hydroxylase, which is necessary for vitamin D synthesis).
Therefore, hyperparathyroidism would result in inappropriate osteoclastic activity that would lead to bone destruction.
There are also several important neoplastic lesions of bone that every student should be relatively familiar with. Note that those classified as benign aggressive tumors have a propensity to undergo malignant transformation:
- Osteoid osteoma
- Fibrous cortical defect
- Giant cell tumors
- Ewing’s osteosarcoma
- Ewing’s of tubular bones
- Metastatic disease