Bone

Bone is the hardest tissue of the human body, and is second only to cartilage in its ability to withstand stress. Bone is a specialized form of connective tissue, which, like other forms of connective tissue, consists of cells and intercellular material. The feature that distinguishes bone from other tissues is the mineralization of its matrix. Over 90% of the matrix is Type I collagen. Packed in and around these fibers are small crystallites of a calcium phosphate salt called hydroxyapatite. This is what makes bone hard. If we remove the crystallites chemically, the bone retains its structure. It still looks like bone, but now it has been decalcified and is quite soft In distinction to cartilage, there are always blood vessels running in and around bone. This is really necessary since calcification of the matrix greatly impedes diffusion of nutrients through it.

Classification of bones.

On the basis of shape, bones can be classified into four groups: long, short, flat and irregular. Long bones (e.g. tibia and metacarpals) are longer in one dimension and consist of a shaft and two ends. Short bones are nearly equal in length, depth and width (e.g. carpals/bones of the wrist). Flat bones are thin and platelike (e.g. bones of the skull) while irregular bones have a complex shape (e.g. vertebrae).

Types of bone.

From a gross point of view, bone can be divided into two classes, compact or spongy. These are well shown in a typical long bone such as the femur which consists of three main parts: 1) the head or epiphysis, 2) the neck or metaphysis and 3) the shaft or diaphysis. If you slice a bone longitudinally, the differences between compact and spongy bone are visible to the naked eye. The diaphysis looks solid, practically without holes or gaps--in other words, it is compact. The metaphysis and epiphysis on the other hand are composed of thousands of spicules or trabeculae of bone, interconnected like a sponge--hence the name spongy bone.

The trabeculae of spongy bone follow the lines of principal stress to which a bone is subjected. If the lines of principal stress are altered--for example when a joint or fracture becomes fused in an abnormal position--the cells of bone immediately set about constructing an entirely new arrangement of trabeculae, better suited to the new conditions. Compact bone may increase or decrease in amount, according to functional demands placed upon it.

Bone is also classified at the microscopic level. Here we may distinguish woven (primary) and lamellar (secondary) bone. Woven bone occurs in the embryo and fetus, and in healing fractures--whenever there is a need for bone in a hurry. It is a temporary expedient, formed in a relatively sloppy fashion. The matrix is composed of coarsely interlaced collagen fibers, with an abundance of ground substance, so that it stains darkish-blue with hematoxylin. It has more osteocytes, scattered in its matrix than does lamellar bone. It is replaced in adults by lamellar bone, except in a very few places in the body, (e.g. near the sutures of the flat bones of the skull, in tooth sockets and in the insertions of some tendons). Lamellar bone is formed more slowly and carefully by the successive laying down of layers (lamellae) of fine collagenous bundles, highly oriented in parallel fashion, with relatively less ground substance, giving the matrix a pinker color with H & E. The osteocytes are more widely spaced, oriented parallel with the lamellae and are almond-shaped with long processes.

Remodeling of compact bone goes on throughout life, even after growth has ceased.

Bone cells.

The cells of bone are of four main types: 1) osteoprogenitors 2) osteoblasts, 3) osteocytes, and 4) osteoclasts,

Osteoprogenitor cells are pale-staining, spindle-shaped cells found in the vicinity of bone surfaces such as the periosteum, endosteum and lining Haversian canals. They are derived from mesenchyme. They are happy cells, because their job is reproduction. Their offspring may continue to reproduce, or they may specialize as osteoblasts, depending upon the local conditions, and the need for bone formation.

Osteoblasts are derived from osteoprogenitor cells. They are bone-forming cells, and are located on the surface of newly forming bone. These are pear-shaped cells with a large spherical nucleus at the smaller end, away from the bone. The cytoplasm is strongly basophilic, indicative of a cell which is actively synthesizing protein. It has a prominent Golgi body, indicative of secretory function. Osteoblasts are responsible for the synthesis of the organic components of bone matrix--type I collagen, glycosaminoglycans and glycoproteins--as well as for the later deposition of the inorganic components of bone. Collagen constitutes 90% of the organic matrix. These cells are exclusively located on the surface of bone, where they secrete a coating (osteoid) over bone in regions of bone formation, or osteogenesis. Osteoid is not bone, as it has not yet been mineralized. It can be thought of as ‘pre-bone’. Osteogenesis always takes place on bone surfaces, never interstitially as in cartilage. Osteoblasts are in contact with one another through thin cytoplasmic processes. As they secrete bone matrix, they become trapped within it and then reside in lacunae within the bone. These trapped osteoblasts are then called osteocytes.

Osteocytes. Like the osteoblasts from which they are derived, osteocytes are also connected by cytoplasmic extensions which pass through thin channels in the bone, called canaliculi--these friendly cells are holding hands with one another. The canalicular system provides a series of minute channels for circulating fluids through the dense, calcified matrix. Osteocytes appear flattened and unremarkable under the light microscope, but they are actively engaged in the maintenance of the bone matrix. They secrete substances necessary for bone maintenance. Death of the osteocytes is followed by resorption of the bone by osteoclasts

Osteoclasts are bone-resorbing cells. These are large, motile, multinucleated cells which are formed by the fusion of monocytes from the blood. Osteoclasts do not bite off the bone and chew it up like a potato chip. Instead they secrete lactic and citric acid, collagenase, and other proteolytic and proteoglycan degrading enzymes that attack the bone matrix, dissolve the calcified ground substance, and are actively engaged in the elimination of debris formed during the resorption of bone. When active, the osteoclast rests directly on the surface of the bone which is being resorbed. As a result of the resorption activity, a shallow bay, called Howship's lacuna, is formed directly under the osteoclast. Osteoclasts can rapidly destroy bone of any kind, alive or dead, compact or spongy, woven or lamellar, new or old. This allows for constant remodeling of bone throughout life.

Bone mineralization.

Within the cytoplasm of osteoblasts which are sitting on growing bone surfaces, the specialized metabolic machinery serves to construct the complex molecules of collagen and ground substance, then secretes them onto the surface of the adjacent bone. Note that what has been secreted is not bone; it is merely bone matrix since it has not yet been calcified. Uncalcified bone matrix is called osteoid. The final step in osteogenesis, then, is calcification of the matrix. Calcification involves the formation within the matrix of numerous crystallites of hydroxyapatite [Ca₁₀(PO₄)₆(OH)₂]. Membrane bound "matrix vesicles" are secreted by the osteoblasts. These tiny vesicles, about 100nm in diameter, contain alkaline phosphatase and other enzymes, and are loaded with calcium and phosphate ions. The loaded matrix vesicles rupture, effecting an increase in the local concentration of minerals sufficient to initiate mineralization. Hydroxyapatite crystals are then deposited within and at the surfaces of these vesicles. The hydroxyapatite crystals are arranged in an ordered array along the length of the collagen fibers. The small centers of mineralization grow and eventually fuse with each other—thus the mineralization spreads over the entire bone. Ground substance surrounds and stabilizes these crystals. This interaction of hydroxyapatite with collagenous fibers and proteoglycans brings about the hardness and rigidity of bone. The continual remodeling of bone throughout life assures that the bone never becomes fully mineralized and thus relatively inert metabolically. Over 90% of the calcium and phosphate in the body is in the bone.

Structure of Bone

As we have seen, bone can be classified microscopically as woven or lamellar. Woven bone has a "messy" appearance, with osteocytes randomly scattered in matrix which is richer in ground substance, and devoid of the lamellae which are characteristic of mature (lamellar) bone. However, woven bone is converted into lamellar bone, and lamellar bone itself is constantly remodeled, even after growth has ceased. As we will see in the lab, lamellar bone is laid down on the surface of woven bone. However woven bone is also replaced, and compact bone is remodeled, by the formation of Haversian systems or osteons in the existing bone. How this happens is quite fascinating. Blood vessels with associated cells, especially osteoclasts, progressively erode a tube about 200 microns in diameter through pre-existing bone. This results in a resorption cavity. Osteoprogenitor cells are carried into the tube by the blood vessels and differentiate into osteoblasts. When the resorption cavity has reached some determined diameter, enlargement ceases and osteoblasts begin to lay down concentric layers of lamellar bone on the surface of the cavity. The collagen fibers of each lamella lie parallel to one another; however the fibers in adjacent lamellae are laid down at different angles to one another. This adds to the strength of the bone. Ultimately the tube is narrowed to a tiny canal, containing the blood vessel, nerves and the sparse connective tissue of the endosteum. This is called an Haversian canal, which together with the associated lamellae of bone make up an Haversian system. These Haversian systems are interconnected by lateral tunnels called Volkmann's canals, through which blood vessels pass.

As the osteoblasts lay down matrix, they are trapped in it, and become osteocytes which remain interconnected with one another through the canaliculi. Canaliculi also extend into the Haversian canal, so osteocytes can transport nutrients from the blood vessel in the Haversian canal to other osteocytes deeper in the bone. Surrounding the outer border of each osteon is a "cement line", a layer of mineralized matrix which is deficient in collagen fibers. The cement lines develop when bone formation follows a phase of bone removal or resorption.

Bone is constantly remodeled by the formation of new osteons. These new osteons may totally or partially replace existing osteons, depending upon where a blood vessel begins to erode a tube. As the new Haversian system is constructed, the concentric lamellae of a pre-existing osteon may be partly destroyed, leaving short interstitial lamellae.

A third class of lamellae is found on the inner and outer surfaces of bone shafts. These may be several layers thick and are called circumferential lamellae. They are created by the ordered synthesis of bone on the surface of pre-existing bone. In this way, weight-bearing bones can become thicker and stronger.

Finally, the surfaces of bone are covered by either a periosteum (external) or an endosteum (internal). The periosteum is a layer of dense connective tissue, and is composed of an outer fibrous layer and an inner cell-rich layer containing osteoprogenitor cells and osteoblasts (if bone formation is in progress). The endosteum is much thinner than the periosteum and consists of osteoprogenitor cells and a small amount of connective tissue. Osteoclasts may be present if bone resorption is in progress. This layer is usually lost during tissue preparation. The primary functions of these tissues are nutrition of the bone and to provide a continuous supply of new osteoblasts (from osteoprogenitor cells) for repair or growth of bone.

During the early growth of the individual, the medullary cavity of long bones, and the spaces in the spongy bone, contain red bone marrow. This is the tissue in which red blood cells develop. In the later stages of growth, and in the adult, when the rate of blood cell formation has diminished, the tissue of the medullary cavity consists mostly of fat cells; it is then called yellow marrow. Under the appropriate stimuli, the yellow marrow can revert to red marrow. The spaces of the spongy bone generally retain the red marrow and continue to function in blood cell formation throughout life.

Blood supply.

The blood supply to long bones comes from three main sources: 1) The nutrient artery penetrates the diaphysis (shaft) and branches in both directions, lateral branches supplying about 2/3 of the thickness of the shaft, as well as the marrow sinusoids. In the metaphysis (neck), the vessels form capillary loops at the site of cartilage invasion. 2) The periostal arteries penetrate the metaphysis and contribute to the capillary loops at the site of cartilage invasion. 3) The epiphyses are supplied by the main epiphyseal arteries.

Bone Formation. (Ossification)

The construction of bone as a functional organ is a complex process. Bone constantly enlarges and renews itself during development, and at the same time adapts to provide support, protection, mechanical strength and hemopoietic activities. The formation of functionally competent bone is the culmination of several integrated processes: (1) intramembranous ossification; (2) endochondral ossification; (3) growth; (4) modeling of bone into its desired shape: (5) the constant replacement of old bone by remodeling.

In the histogenesis of bone, the first bone tissue formed is an immature type known as primary or woven bone. This is a temporary bone that is soon replaced by secondary or lamellar bone.

Bone formation is classified as endochondral or intramembranous, depending upon whether a cartilage model serves as the precursor of bone (endochondral bone formation) or whether bone is formed de novo from condensed mesenchymal tissue, without the use of a cartilage precursor (intramembranous bone formation). The bones of the extremities, and those parts of the skeleton which bear weight (e.g. vertebrae) develop by endochondral bone formation. The flat bones of the skull, the mandible and the maxilla develop by intramembranous bone formation. In both processes, the bone that first appears is primary or woven bone, but it is soon replaced by secondary or lamellar bone. The replacement bone is laid down on the preexisting bone by appositional growth and is identical in both cases. Additionally, both woven bone and lamellar bone are constantly remodeled, either by the action of osteoclasts followed by the deposition of lamellar bone on the surface of pre-existing bone, or by the development of Haversian systems in pre-existing bone.

Intramembranous bone formation.

Most flat bones are formed by the process of intramembranous bone formation. Intramembranous ossification (bone formation) occurs within an area of well vascularized, primitive mesenchymal tissue. (No cartilage is necessary). In the mesenchymal condensation layer, the starting point for ossification is called the primary ossification center. A cluster of mesenchymal cells differentiates into osteoblasts, which begin to secrete osteoid. As the spicules of osteoid thicken, osteoblasts are trapped and become osteocytes. At this stage of the ossification, the spicules are thin and needle-like; however they are lengthened and thickened by continual apposition of osteoid, and mineralization of the matrix by osteoblasts. Eventually this increased growth results in fusion of the primary ossification centers. In regions of woven bone which are destined to become compact bone, the spaces between the spicules are filled by continued deposition of bone. This compact bone is eventually remodeled. In regions of woven bone which are destined to persist as spongy bone, the thickened plates of bone are penetrated by blood vessels and undifferentiated mesenchymal tissue, giving rise to bone marrow cells. These spaces, the diploe, will later become an important site of hemopoiesis. As with all bones, the final product is surrounded by a periosteum and endosteum.

Endochondral bone formation.

Most long bones are formed by endochondral ossification. In this process a small cartilagenous model of the bone is first constructed, then replaced by bone tissue. Early in development, the mesenchyme in the region of the future bone differentiates into hyaline cartilage. As the cartilage grows, both interstitially and by apposition on its surface, the shape of a long bone can be seen. With growth of the cartilage model, the perichondrium is invaded by capillaries, triggering the transformation of the perichondrium into a periosteum. The innermost cells of the periosteum, exposed to these new environmental conditions, specialize as osteoblasts and lay down a bone collar around the middle of the shaft. (Note: Periosteal bone is in fact intramembranous bone, since it develops from connective tissue, and not on a spicule of calcified cartilage).

With the development of the bone collar, diffusion of nutrients into the cartilage is prevented and the chondrocytes at the center of the shaft (diaphysis) start to swell up (hypertrophy), and soon disintegrate and die. Simultaneously the matrix surrounding these dying cells begins to calcify. Small blood vessels now penetrate the bone collar through holes bored by osteoclasts, and invade the area of calcified cartilage. Osteoprogenitor cells, which are carried in with the blood, differentiate into osteoblasts and begin to lay down bone on the calcified cartilage. In this manner, what was cartilage, becomes a primary ossification center built of spongy bone, constructed on a framework of calcified cartilage matrix. As soon as this bone is formed, it begins to be resorbed by the action of osteoclasts, resulting in the formation of a hollow marrow cavity. The repetition of this process in both directions soon results in the formation of a marrow cavity filling the diaphysis of the bone, with only a few of the original trabeculae remaining. At the same time, the bone collar is thickened by apposition of bone on its outer surface, and widened by resorption of bone on its inner surface. At this point, the head (epiphysis) at each end of the bone is still composed of cartilage, while the shaft consists of a collar of bone around the marrow cavity.

In the later stages of embryonic development, a similar process takes place in the epiphyses at each end of the bone, although not simultaneously. Again, chondrocytes hypertrophy and die, the surrounding matrix calcifies, blood vessels and osteoprogenitor cells invade, and bone is laid down on the trabeculae of calcified cartilage, which are soon resorbed by the action of osteoclasts. This is called a secondary ossification center. However, no bone collar forms on the surface of the cartilage which forms the epiphysis, as this is specialized as articular cartilage. The function of these secondary ossification centers is to allow the ends of the bone to expand radially, rather than longitudinally. Thus the surface of the joint retains it size relative to the increase in size of the bone and the individual.

By now, all that remains of the original cartilagenous model is the articular cartilage which covers each of the epiphyses, and two strips of cartilage (epiphyseal growth plates) at the junctions between the diaphysis and epiphysis (i.e at the metaphysis). Articular cartilage plays no role in the future growth of the bone. The epiphyseal growth plates are solely responsible for the subsequent increase in length of the bones. Increase in width of the bone is achieved by the apposition of new bone on the outer surface, beneath the periosteum.

Growth in length of the bones, and therefore growth in length of the individual, is determined by the activity of the cartilage cells living in the epiphyseal growth plates. It is here that growth hormone, coming from the pituitary gland, exerts an important effect. Too much hormone yields gigantism; too little, dwarfism.

It is important to understand how the multiplication, growth and death of the chondrocytes in the growth plates result in an increase in length of the bone. Five zones are distinguished within the growth plate, based on changes in the activity and appearance of the chondrocytes. Note: These zones are not discrete, they blend into one another

1). On the epiphyseal side, the zone of reserve cartilage consists of normal-appearing hyaline cartilage, with chondrocytes embedded in matrix.

2). In the zone of proliferation, chondrocytes start to divide rapidly and form longitudinally arranged columns of cells. Many mitotic figures can be seen, and the cells synthesize and secrete matrix. This zone is the driving force behind bone elongation.

3). Towards the bottom of the proliferative zone, the cells enter the zone of hypertrophy, where they swell up due to the intake of water, and also accumulate glycogen. The matrix between chondrocytes is squeezed into thin vertical and horizontal septae, due to the enlargement of the cells and their lacunae.

4). In the zone of calcification, the hypertrophied cells begin to disintegrate and die, and the septae of hyaline matrix between the last three or four disintegrating cells suddenly start to calcify, due to the deposition of crystals of hydroxyapatite.

5). In the zone of ossification, some of the calcified cartilage at the tip of the septae is removed by the action of osteoclasts. The remainder of these septae become scaffolds for the deposition of bone. This area is constantly invaded by capillaries carrying in osteoprogenitor cells which specialize as osteoblasts, which deposit a thin layer of osteoid on the calcified cartilage that has escaped resorption. The osteoid is subsequently mineralized, as discussed above. In this way, bone spicules are formed with a central area of calcified cartilage and a thin covering of primary bone. Thus although the growth cartilage continues to proliferate, it does not grow thicker, since it is continually eroded and replaced by spongy bone on its diaphyseal side. The rates of these two processes (cell proliferation and destruction) are approximately equal, and thus the epiphyseal plate does not increase in thickness. Instead it is progressively displaced towards the epiphyseal end of the bone, resulting in an increase in bone length.

Bone growth and remodeling

At the same time that long bones are growing longer, they are also growing thicker, and thus there has to be a constant remodeling in order to maintain the shape of the bone. This occurs by deposition of bone on the external surface of the bone collar, and resorption of bone on the internal surface. Thus the bone becomes thicker, and the size of the marrow cavity increases. At the metaphysis, the opposite occurs--bone is deposited on the internal surface and removed on the external surface, thus allowing the shaft to remain round. Eventually, growth of the cartilage in the epiphyseal plate ceases, and the cartilage is replaced by bone. At this point, the epiphysis and diaphysis become continuous in a network of spongy bone, and any increase in the length of the bone ceases (at about age 20). Finally, the shaft is finished off by the slow addition of layers of lamellar bone on both periosteal and endosteal surfaces. This surface lamellar bone constitutes the inner and outer circumferential lamellae. Thus the shaft of a typical long bone from a young adult may be seen in cross section, from outside inward with 1) periosteum, 2) outer circumferential lamellae 3) osteons (both mature and actively forming) and interstitial lamellae, 4) inner circumferential lamellae, 5) endosteum.

Joints

All bones are connected by joints to form the skeleton. Some of these joints allow free movement of the bone (diarthroses), while others allow only limited movement (synarthroses). Diarthroses are usually found between the long bones of the skeleton. The articulating surfaces are capped with hyaline cartilage and surrounded by an articular capsule which is filled with synovial fluid. The capsule is a continuation of the connective tissue surrounding the bone, and consists of an outer fibrous layer, and an inner synovial membrane, which forms the lining of the joint cavity. The synovial membrane is not covered by an epithelium; instead the surface facing the fluid within the joint is merely a specialized connective tissue, supplied with blood vessels, lymphatics and nerves. The cells of this membrane secrete the synovial fluid. This lubricating and shock-absorbing fluid is yellowish and viscid, containing cellular debris, mucin and glycoproteins.

Calcium homeostasis.

Because the skeleton contains 99% of the calcium in the body, it is not surprising that bone tissue acts as a calcium reservoir. The normal blood level of calcium is about 10 mg %, and is maintained within 1 mg % of this level. Under normal conditions, this level is maintained by a continuous exchange of calcium ion between bone tissue and the blood. This exchange occurs between the hydroxyapatite crystals of calcified lamellae and blood. When calcium levels drop in the blood, calcium is released from bone, thus maintaining the level in the blood. The two most important hormones that are involved in calcium homeostasis are parathyroid hormone (PTH) and calcitonin.When calcium levels drop in the blood, the parathyroid releases PTH, which has a number of different actions. In bone it increases the activity of osteoclasts, thus speeding up bone resorption, with the subsequent release of calcium. In the kidney it increases the reabsorption of calcium from the forming urine. If the calcium level increases too much in response to the action of PTH, calcitonin comes into play. This hormone, which is secreted by the parafollicular cells of the thyroid, inhibits bone resorption by inhibiting the activity of osteoclasts. Since the maintenance of blood calcium levels is so critical to the functioning of numerous biological processes such as muscle contraction, nerve conduction, blood clotting, secretion, these two hormones play a crucial role in homeostasis.

Rickets (which occurs in children) and osteomalacia (which occurs in adults) are both characterized by the deposition of matrix which fails to calcify. In children this is usually brought about by a deficiency of vitamin D. In adults it may also arise from kidney disease as well as decreased intestinal absorption of calcium and vitamin D: it produces an increased fragility of the bones. In children, the growth cartilage is grossly thickened, and skeletal deformities may arise in the weakened bones. In osteoporosis, which is frequently found in immobilized patients and postmenopausal women, there is a decrease in bone mass caused by decreased bone formation, increased bone resorption or both. This results in an increased fragility of the bones. Osteoporosis can be fairly effectively treated by taking a combination of estrogen and progesterone (hormone replacement therapy/HRT). However, a number of studies have suggested that HRT increases the risk of developing breast cancer.