FORMATION OF BONE All bones are of mesodermal origin. The process of bone formation is called ossification. We have seen that formation of most bones is preceded by the formation of a cartilaginous model, which is subsequently replaced by bone. This kind of ossification is called endochondral ossification; and bones formed in this way are called cartilage bones. In some situations (e.g., the vault of the skull) formation of bone is not preceded by formation of a cartilaginous model. Instead bone is laid down directly in a fibrous membrane. This process is called intramembranous ossification; and bones formed in this way are called membrane bones. The bones of the vault of the skull, the mandible, and the clavicle are membrane bones. Intramembranous Ossification The various stages in intramembranous ossification are as follows: 1.At the site where a membrane bone is to be formed the mesenchymal cells become densely packed (i.e., a mesenchymal condensation is formed). 2.The region becomes highly vascular. 3.Some of the mesenchymal cells lay down bundles of collagen fibres in the mesenchymal condensation. In this way a membrane is formed. 4.Some mesenchymal cells (possibly those that had earlier laid down the collagen fibres) enlarge and acquire a basophilic cytoplasm, and may now be called osteoblasts (Fig. 1A). They come to lie along the bundles of collagen fibres. These cells secrete a gelatinous matrix in which the fibres get embedded. The fibres also swell up. Hence the fibres can no longer be seen distinctly. This mass of swollen fibres and matrix is called osteoid (Fig.1B). 5.Under the influence of osteoblasts calcium salts are deposited in osteoid. As soon as this happens the layer of osteoid can be said to have become one lamellus of bone (Fig. 1C). 6.Over this lamellus, another layer of osteoid is laid down by osteoblasts. The osteoblasts move away from the lamellus to line the new layer of osteoid. However, some of them get caught between the lamellus and the osteoid (Fig. 1D). The osteoid is now ossified to form another lamellus. The cells trapped between the two lamellae become osteocytes (Fig. 1D). 7.In this way a number of lamellae are laid down one over another, and these lamellae together form a trabeculus of bone (Fig. 1E). The first formed bone may not be in the form of regularly arranged lamellae. The elements are irregularly arranged and form woven bone. -1-
A B C D E Fig. 10: Scheme to show how bony lamellae are laid down over one another (Schematic representation) Endochondral Ossification The essential steps in the formation of bone by endochondral ossification are as follows: 1. At the site where the bone is to be formed, the mesenchymal cells become closely packed to form a mesenchymal condensation (Fig. 2A). 2. Some mesenchymal cells become chondroblasts and lay down hyaline cartilage (Fig.2B). Mesenchymal cells on the surface of the cartilage form a membrane called the perichondrium. This membrane is vascular and contains osteoprogenitor cells. 3. The cells of the cartilage are at first small and irregularly arranged. However, in the area where bone formation is to begin, the cells enlarge considerably (Fig.2C). 4. The intercellular substance between the enlarged cartilage cells becomes calcified, under the influence of alkaline phosphatase, which is secreted by the cartilage cells. The nutrition -2-
A B C D E F G H I Fig. 2: Endochondral ossification. Formation of cartilaginous model (Schematic representation) -3-
to the cells is thus cut off and they die, leaving behind empty spaces called primary areolae (Fig. 2C). 5.Some blood vessels of the perichondrium (which may be called periosteum as soon as bone is formed) now invade the cartilaginous matrix. They are accompanied by osteoprogenitor cells. This mass of vessels and cells is called the periosteal bud. It eats away much of the calcified matrix forming the walls of the primary areolae, and thus creates large cavities called secondary areolae (Fig. 2D). 6.The walls of secondary areolae are formed by thin layers of calcified matrix that have not dissolved. The osteoprogenitor cells become osteoblasts and arrange themselves along the surfaces of these bars, or plates, of calcified matrix (Fig. 2E). 7.These osteoblasts now lay down a layer of ossein fibrils embedded in a gelatinous ground substance (i.e., osteoid), exactly as in intramembranous ossification (Fig. 2F). This osteoid is calcified and a lamellus of bone is formed (Fig. 2G). 8.Osteoblasts now lay down another layer of osteoid over the first lamellus. This is also calcified. Thus two lamellae of bone are formed. Some osteoblasts that get caught between the two lamellae become osteocytes. As more lamellae are laid down bony trabeculae are formed (Fig. 2H). 9.It may be noted that the process of bone formation in endochondral ossification is exactly the same as in intramembranous ossification. The calcified matrix of cartilage only acts as a support for the developing trabeculae and is not itself converted into bone. 10.At this stage the ossifying cartilage shows a central region where bone has been formed. As we move away from this area we see: 1.A region where cartilaginous matrix has been calcified and surrounds dead and dying cartilage cells 2.A zone of hypertrophied cartilage cells in an uncalcified matrix 3.Normal cartilage in which there is considerable mitotic activity. In this way formation of new cartilage keeps pace with the loss due to replacement by bone. The total effect is that the ossifying cartilage progressively increases in size. Conversion of Cancellous Bone to Compact Bone All newly formed bone is cancellous. It is converted into compact bone as follows. Each space between the trabeculae of cancellous bone comes to be lined by a layer of osteoblasts. The osteoblasts lay down lamellae of bone as already described. The first lamellus is formed over the inner wall of the original space and is, therefore, shaped like a ring. Subsequently, concentric lamellae are laid down inside this ring thus forming an osteon. The original space becomes smaller and smaller and persists as a Haversian canal. The first formed Haversian systems are called atypical Haversian systems or primary osteons. These osteons do not have a typical lamellar structure, and their chemical composition may also be atypical. Primary osteons are soon invaded by blood vessels and by osteoclasts that bore a new series of spaces through them. These new spaces are again filled in by bony lamellae, under the influence of osteoblasts, to form secondary osteons (or typical Haversian systems). The process of formation and destruction of osteons takes place repeatedly as the bone enlarges in size; and continues even after birth. In this way the internal structure of the bone can be repeatedly remodelled to suit the stresses imposed on the bone. -4-
Interposed in between osteons of the newest series there will be remnants of previous generations of osteons. The interstitial lamellae of compact bone represent such remnants. When a newly created cavity begins to be filled in by lamellae of a new osteon, the first formed layer is atypical in that it has a very high density of mineral deposit. This layer can subsequently be identified as a cement line that separates the osteon from previously formed elements. As the cement line represents the line at which the process of bone erosion stops and at which the process of bone formation begins, it is also called a reversal line. From the above it will be clear why cement lines are never present around primary osteons, but are always present around subsequent generations of osteons. HOW BONES GROW A hard tissue like bone can grow only by deposition of new bone over existing bone i.e., by apposition. We will now consider some details of the method of bone growth in some situations. Growth of Bones of Vault of Skull In the bones of the vault of the skull (e.g., the parietal bone) ossification begins in one or more small areas called centres of ossification and forms following the usual process. At first it is in the form of narrow trabeculae or spicules. These spicules increase in length by deposition of bone at their ends. As the spicules lengthen they radiate from the centre of ossification to the periphery. Gradually the entire mesenchymal condensation is invaded by this spreading process of ossification and the bone assumes its normal shape. However, even at birth the radiating arrangement of trabeculae is obvious. The mesenchymal cells lying over the developing bone differentiate to form the periosteum. The embryonic parietal bone, formed as described above, has to undergo considerable growth. After ossification has extended into the entire membrane representing the embryonic parietal bone, this bone is separated from neighbouring bones by intervening fibrous tissue (in the region of the sutures). Growth in size of the bone can occur by deposition of bone on the edges adjoining sutures (Figs.3,4). Growth in thickness and size of the bone also occurs when the overlying periosteum forms bone (by the process of intramembranous ossification described above) over the outer surface of the bone. Simultaneously, there is removal of bone from the inner surface. In this way, as the bone grows in size, there is simultaneous increase in the size of the cranial cavity. Development of a Typical Long Bone In the region where a long bone is to be formed the mesenchyme first lays down a cartilaginous model of the bone (Figs. 5 A to C). This cartilage is covered by perichondrium. Endochondral ossification starts in the central part of the cartilaginous model (i.e., at the centre of the future shaft). This area is called the primary centre of ossification (Fig. 5D). Gradually, bone formation extends from the primary centre towards the ends of shaft. This is accompanied by progressive enlargement of the cartilaginous model. Soon after the appearance of the primary centre, and the onset of endochondral ossification in it, the perichondrium (which may now be called periosteum) becomes active. The osteoprogenitor cells in its deeper layer lay down bone on the surface of the cartilaginous -5-
A B C D Fig. 3: Scheme to show how skull bones grow model by intramembranous ossification. This periosteal bone completely surrounds the cartilaginous shaft and is, therefore, called the periosteal collar (Figs. 5D and E). The periosteal collar is first formed only around the region of the primary centre, but rapidly extends towards the ends of the cartilaginous model (Figs. 5F and G). It acts as a splint, and gives strength to the cartilaginous model at the site where it is Fig. 4: Growth of skull bones at sutures weakened by the formation of secondary areolae. We shall see that most of the shaft of the bone is derived from this periosteal collar and is, therefore, membranous in origin. At about the time of birth the developing bone consists of a part called the diaphysis (or shaft), that is bony, and has been formed by extension of the primary centre of ossification, and ends that are cartilaginous (Fig. 5F). At varying times after birth secondary centres of endochondral ossification appear in the cartilages forming the ends of the bone (Fig. 5G). -6-
A B C D E F G H Fig. 5: Formation of a typical long bone (Schematic representation) -7-
These centres enlarge until the ends become bony (Fig. 5H). More than one secondary centre of ossification may appear at either end. The portion of bone formed from one secondary centre is called an epiphysis. For a considerable time after birth the bone of the diaphysis and the bone of any epiphysis are separated by a plate of cartilage called the epiphyseal cartilage, or epiphyseal plate. This is formed by cartilage into which ossification has not extended either from the diaphysis or from the epiphysis. We shall see that this plate plays a vital role in growth of the bone. Growth of a Long Bone A growing bone increases both in length and in girth. The periosteum lays down a layer of bone around the shaft of the cartilaginous model. This periosteal collar gradually extends to the whole length of the diaphysis. As more layers of bone are laid down over it, the periosteal bone becomes thicker and thicker. However, it is neither necessary nor desirable for it to become too thick. Hence, osteoclasts come to line the internal surface of the shaft and remove bone from this aspect. As bone is laid down outside the shaft it is removed from the inside. The shaft thus grows in diameter, and at the same time its wall does not become too thick. The osteoclasts also remove trabeculae lying in the centre of the bone that were formed by endochondral ossification. In this way, a marrow cavity is formed. As the shaft increases in diameter there is a corresponding increase in the size of the marrow cavity. This cavity also extends towards the ends of the diaphysis, but does not reach the epiphyseal plate. Gradually most of the bone formed from the primary centre (i.e., of endochondral origin) is removed, except near the bone ends, so that the wall of the shaft is ultimately made up entirely of periosteal bone formed by the process of intramembranous ossification. To understand how a bone grows in length, we will now take a closer look at the epiphyseal plate. Depending on the arrangement of cells, three zones can be recognized: (Fig. 6). 1.Zone of resting cartilage. Here the cells are small and irregularly arranged. 2.Zone of proliferating cartilage. This is also called the zone of cartilage growth. In this zone the cells are larger, and undergo repeated mitosis. As they multiply, they come to be arranged in parallel columns, separated by bars of intercellular matrix. Fig. 6: Structure of an epiphyseal plate (Schematic representation) -8-
3.Zone of calcification. This is also called the zone of cartilage transformation. In this zone the cells become still larger and the matrix becomes calcified. Next to the zone of calcification, there is a zone where cartilage cells are dead and the calcified matrix is being replaced by bone. Growth in length of the bone takes place by continuous transformation of the epiphyseal cartilage to bone in this zone (i.e., on the diaphyseal surface of the epiphyseal cartilage). At the same time, the thickness of the epiphyseal cartilage is maintained by the active multiplication of cells in the zone of proliferation. When the bone has attained its full length, cells in the cartilage stop proliferating. The process of calcification, however, continues to extend into it until the whole of the epiphyseal plate is converted into bone. The bone substance of the diaphysis and that of the epiphysis then become continuous. This is called fusion of the epiphysis. Metaphysis The portion of the diaphysis adjoining the epiphyseal plate is called the metaphysis. It is a region of active bone formation and, for this reason, it is highly vascular. The metaphysis does not have a marrow cavity. Numerous muscles and ligaments are usually attached to the bone in this region. Even after bone growth has ceased, the calcium turnover function of bone is most active in the metaphysis, which acts as a store house of calcium. The metaphysis is frequently the site of infection (osteomyelitis) because blood vessels show hairpin bends and blood flow is sluggish. Added Information -9-