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السبت، 7 يناير 2012

Histology slide Endochondral Ossification


Endochondral Ossification

All of the long bones and many others of the body, are preformed embryologically in hyaline cartilage and then replaced by bone by endochondral ossification. Such a change has begun in the middle of the shaft of this bone, thanks to the invasion of blood vessels and their accompanying primitive connective tissue. Pale pink cartilage is seen in the head of the bone. A dark pink periosteal bone collar has already formed around the middle of the shaft, and ossification is proceeding toward both ends of the cartilage model. The dark pink strands lying outside the whole bone are dense collagenous tissue of periosteum (around the bony part) and perichondrium (around the cartilaginous part). H & E stain.

Endochondral ossification in greater detail. The cartilage cells (chondrocytes) near the region of active ossification have enlarged (hypertrophied) and lined up more or less in columns. The purplish material in the center of the shaft is primitive bone marrow, with reticular cells and developing blood cells. The vascular elements of the marrow tissue actively invade the cartilage above, leaving spicules of calcified cartilage, upon which bony matrix will be deposited. The dark pink spicules here are made of bone; the paler pink, small spicules at the leading edge of the cartilage are made of calcified cartilage.

Endochondral ossification in Mallory stain. Cartilage is light blue and bone is dark blue. A thin layer of bone has already been laid down on the surface of the cartilage spicules along the leading edge of cartilage. Blood cells in the marrow cavity are red. The very dark blue at the lower left and right is spongy bone of the periosteal bone collar of the shaft. This will later be remodeled into Haversian systems of compact bone.

Head of fetal bone still made of hyaline cartilage. Near the point where ossification is going on (upper right corner) the cartilage cells become larger and the cartilage matrix becomes calcified (purple instead of pale pink here, as stained in H & E). A small amount of dark pink bone has been laid down on the surface of the calcified cartilage. Later on, a secondary center of ossification will form in the head of the bone, and the cartilage that remains between the two centers of ossification will be the epiphyseal plate for growth of the bone in length.

Region of ossification at higher magnification -- same stain as previous slide. Chondrocytes are hypertrophied, degenerating, and lined up in columns at the right. As the marrow tissue invades the cell columns, spicules of cartilage will be left. The cartilage matrix is calcified (purple), and one small area of bone deposition, has begun on it (the red color at the upper right). The small cells caught in the red matrix are osteocytes.

Another detail of ossification. Calcified cartilage spicules are purple-blue; bone deposits are purple-red. Gradually the cartilaginous portions will be resorbed as the bone is constantly reshaped, until finally there will be no trace of cartilage left. The main purpose of the cartilage in the first place was to provide a framework upon which bone deposition could begin.

Spicules showing early endochondral ossification. In H & E stain, the centers of the spicules show the purple of calcified cartilage; the edges are pink because of the bony matrix laid down upon the cartilage.

Spicules of spongy bone (bright red) surrounded by a whole line-up of osteoblasts. The osteoblasts that have previously been trapped in their own salt deposits now lie in lacunae within the spicule and are called osteocytes. The cells of the primitive bone marrow lie between bone spicules.

Detail of bony spicule with typically acidophilic (pink in H & E) matrix. Osteoblasts are lined up along its borders, depositing another layer of matrix. Osteocytes lie within lacunae in the spicule.

EM of active osteoblast laying down the fibers and salts of bone. The cytoplasm of the cell is to the left and contains lots of rough endoplasmic reticulum and many mitochondria. In the lower right corner is mineralized bony matrix containing the typical black CaPO4 (apatite) crystals. Between this matrix and the osteoblast lies a pale area of newly secreted pre-bone (or osteoid) which contains collagen fibrils (note their cross-striations) lying in an as yet unmineralized ground substance.

Spicules of spongy bone stained with Mallory stain. Bone stains blue. Note line of osteoblasts along left hand edge.

 

Enlarged area of spongy bone, so called because there are large, irregular spaces of bone marrow intermixed with the bony substance.

The large central space is a resorption area where young compact bone is being actively remodeled. This is an area where osteoclasts are resorbing the bony substance; notice to large multinucleated osteoclasts toward the left of the cavity next to the intact Haversian system that lies in the upper left corner of the field. Later, osteoblasts will differentiate from the primitive reticular tissue in the resorption cavity and will begin to lay down new bony lamellae around the edge of the cavity. As successive lamellae are laid down, the cavity will gradually grow smaller, until eventually a new Haversian system with a narrow central canal will be formed.

Detail of an osteoclast, a giant, multinucleated cell associated with bone resorption. The shallow bay in which it lies is a Howship's lacuna. The osteoclast is now considered to develop from a separate stem cell in the bone marrow.

EM of an osteoclast, with its ruffled border next to the area where bony matrix is being resorbed. The net effect of a ruffled border is to increase the cell surface area for contact with the collagen fibrils and apatite crystals being resorbed.

Haversian systems of decalcified compact bone, mostly cut in cross section here. The one channel cut longitudinally is a Volkmann's canal; these channels run perpendicular to both the long axis of the bone and the central canals of the Haversian systems.

Decalcified bone with Haversian system cut longitudinally. Notice the central blood vessel and the many concentric bony lamellae around it. As always, osteocytes are trapped in their lacunae.

End of a young long bone, with the pale epiphyseal plate lying between the primary ossification center of the shaft and the secondary ossification center of the head. The plate and the pale area continuous with it, up over the head, are composed of hyaline cartilage. Active ossification is going on along the lower edge of the epiphyseal plate, allowing growth in length of the bone. There is also active ossification along the lower edge of the cartilage that surrounds the head, thus allowing for growth in size of the head of the bone. Bony spicules are seen throughout the centers of ossification, making areas of spongy bone with red marrow between the spicules.

Diagram of a cross-cut chunk of wall of the shaft of a long bone. Most of the substance is compact bone, with Haversian systems cut cross-wise on the uppermost surface and longitudinally on the right-side surface. Volkmann's canals carry blood vessels from the inner and outer bone surfaces to the vessels of the Haversian canals. The lamellae of the Haversian systems are pulled out here so that you can see the lamellar rings. External (or periosteal) circumferential lamellae are seen surrounding the whole bone. Internal (or endosteal) lamellae line the inner surface next to the marrow cavity (to the left). Notice that the inner, endosteal wall bears many spicules of spongy (cancellous, trabecular) bone. The dense collagenous connective tissue coat (the periosteum) looks dark here and surrounds the whole shaft.