Lab 4. Bone & Joints

Objectives

Students should:

understand the histological structure of bone and the differences between compact and spongy bone.
recognize the three characteristic cell types found in bone and understand their functions.
understand the 2 processes by which bone is formed.
understand the remodelling and repair processes seen in bone.

Bone is a type of C.T. in which the intercellular matrix is highly specialized for rigidity and strength. It is a highly dynamic tissue; the structure of mature bone seen in the microscope is the last of numerous cycles in which bony tissue is formed, then resorbed, followed by formation of new bony tissue, etc. Bone is usually formed in layers or lamellae which contain collagen fibers in a nearly parallel array, and a small proportion of proteoglycans and other substances. This organic matrix is soon mineralized by formation of minute apatite-like crystals oriented along the collagen fibers. The collagen fibers of adjacent lamellae are laid down obliquely or perpendicularly. This plywood-like arrangement increases the overall strength.

A specialized layer of dense C.T. called the periosteum covers the outer surface of the bone. The inner surface enclosing the marrow cavity is lined with a very thin, delicate C.T., the endosteum. The cells of the endosteum and the inner layer of the periosteum resemble fibroblasts, but can differentiate into osteoblasts under appropriate stimulation. The principal bone cells are the osteocyte, the osteoblast, and the osteoclast. The osteocyte, or bone cell proper, is found in lacunae lying in or between the lamellae. Very fine channels (canaliculi) containing osteocyte cell processes connect lacunae to each other and to tissue fluid spaces, providing for nutrition and metabolic exchange. The osteocyte is derived from the osteoblast as it becomes surrounded by matrix. The osteoblast is found on the surfaces of bone and is involved with bone deposition. Active sites of bone formation are characterized by large basophilic osteoblasts. The osteoclast is usually found in depressions (Howship's lacunae) at the surfaces of bony tissue and is associated with bone resorption; it is a large multi-nucleated cell derived from blood monocytes.

Bony tissue is classified as either spongy (trabecular, cancellous) or compact bone, depending on the relative proportions of mineralized and soft tissue; most bones contain both spongy and compact regions. Compact bone and the larger trabeculae of spongy bone contain lamellae. In compact bone, however, the predominant structural unit is the osteon, or Haversian system. In the osteon, lamellae are arranged as concentric cylinders surrounding a central Haversian canal. This canal contains nerves, connective tissue and blood vessels. The long axis of an osteon is approximately parallel to the major axis of stress, usually the long axis of the bone. The formation of an osteon can be visualized as a cycle of bone resorption proceeding radially outward from a blood vessel, followed by deposition of new lamellae starting at the outer edge and ending at what becomes a new Haversian canal.

Compact Bone

Those of you with odd boxes should begin with slide #14, described in Section (b) below.

(a) Decalcified bone (slide #25)

Cross-sections of decalcified compact bone appear as an deeply eosinophilic ring surrounding a marrow cavity containing fat cells and blood cells. Surrounding this ring is a layer of C.T., the periosteum which is normally a complete layer although on some slides, only fragments are left. With the diaphragm closed down, look for faint lines throughout the bone. These are lamellae. They will be seen more clearly on slide 14 odd. Just beneath the periosteum, the bone contains several layers of lamellae which run roughly parallel with the surface of the bone (outer circumferential lamellae).

These lamellae may be discontinuous, and are usually present on one side of the bone only. Look for roughly circular arrays of lamellae which are cross-sections through osteons (Haversian systems) and are found in the middle part of the bone tissue, with the Haversian canal surrounded by concentric lamellae. There may be larger spaces within the compact bone filled with a loose C.T. and often lined with basophilic osteoblasts. These are usually areas where bone was previously resorbed and new osteons were in the process of being formed (remodeling). The inner circumferential lamellae form the inner layer of bone adjacent to the marrow cavity. They do not necessarily form continuous layers, and may not be present in all specimens. Volkmann's canals penetrating from the periosteal or endosteal surfaces may be present; they are easily confused with Haversian canals until you recognize that they are oriented roughly at right-angles to the osteon lamellae. Volkmann's canals are tubular passages in the bone through which blood vessels in the periosteum or the marrow cavity send branches to the Haversian canals. On some slides, Volkman's canals may be seen connecting adjacent Haversian canals. The endosteum, which is best seen under high power, is a thin layer of C.T. consisting of a single layer of osteoblasts on the inner margin of the bone.

(b) Ground bone (slide #14, odd boxes)

	Examine the lamellar arrangement and the osteons on a section of undecalcified ground bone. Note: Some slides have both longitudinal and cross-sections which are easily overlooked; check at the edge of the coverslip. Study cross-section first. Osteons have a target-like appearance with the Haversian canal at the center surrounded by concentric lamellae. Blood vessels injected with a red dye are seen. Those in the Haversian canal run parallel to the axis of an osteon, and do not cross lamellae. You may also find blood vessels in Volkmann's canals

	The lamellar arrangement can be discerned under high magnification with the condenser diaphragm set at a small aperture. Observe that within an osteon, adjacent lamellae have different optical properties, alternate lamellae appearing light (clear) and dark (stippled). In the dark lamellae, the collagen fibers are running approximately longitudinally and were cut in cross-section, whereas in the light lamellae, the fibers run approximately circularly and are cut parallel to their axis. Some fibers may be seen running from lamellae to lamellae. In cross-sections, lamellar systems will be seen which do not appear to be complete osteons. These are called interstitial lamellae and are the remains of older bone lamellae which have been only partly resorbed. At the outer edge of an osteon there is a thin layer of cement substance which appears as a refractile (clear) line (cement line). Dark-appearing spaces among the lamellae are lacunae and canaliculi. Lacunae are larger, are mainly oriented parallel to lamellae, and contain osteocytes. The canaliculi appear as fine lines extending radially from the lacunae.

	Study the EMs of an osteocyte sitting in bone matrix (top) and a higher magnification of a cell process extending from an osteocyte through a canaliculus in the bone matrix

Bone Formation

The basic process in bone formation consists of the deposition of a calcifiable organic matrix by osteoblasts, and its subsequent mineralization. In early development, osteoblasts differentiate from mesenchyme cells and from fibroblast-like cells with mesenchymal potentialities. In later development, they differentiate from fibroblast-like osteogenic cells only. Osteoblasts remain relatively stationary while laying down the matrix and become completely embedded in matrix, at which time, they become known as osteocytes.

Bone formation is classified into 2 main types, according to the milieu in which it occurs:

(1) Intramembranous: The bone is formed in and replaces a pre-existing membrane of embryonic connective tissue. A periosteum develops on the surfaces of the newly formed bone, and osteoblasts, which differentiate in it, continue the process of osteogenesis. Bones of the skull and jaw are examples.

(2) Endochondral bone formation: A cartilage model is formed initially; the cartilage is a temporary structure and is eventually destroyed except for the articular surface. Short and long bones are formed this way.

The long bones are formed by a combination of endochondral and intramembranous bone formation. The initial bone collar at the primary center is made by intramembranous ossification. halfway between the ends of the cartilage model, the osteogenic capacity of cells of the perichondrium is activated leading to development of the bone collar. Subsequently, blood vessels penetrate into the cartilage model leading to endochondral ossification. Growth in width of the diaphysis of large bones occurs in a manner similar to that of the "membrane" bones (e.g. calvaria). Most of the true endochondral bone serves merely as a temporary framework which is ultimately resorbed. Mature bone of intramembranous or endochondral origin has essentially the same histological structure, i.e., the lamellar structure just studied.

A non-lamellar form of bone (woven bone) is normally found in rapidly growing areas of embryonic or developing bone, and in healing fractures. Most woven bone is replaced by lamellar bone during remodeling, but woven bone persists at certain sites in the adult (e.g. in tooth sockets, bony sutures, and tendonous and ligamentous insertions). The process of bone remodeling occurs continually in the embryo, fetus and the adult in order to maintain the proper shape of the bone during growth, and for adaptation to normal variations in physiological conditions and to changes in stress throughout life. Remodeling involves the removal of osseous tissue (resorption) in some locations and adding tissue (accretion) in others. In bone resorption, normally both the organic matrix and the mineral crystals are removed simultaneously.

Intramembranous Bone Formation

Examine a section through the skull of a pig fetus, slide #18. The eosinophilic bony spicules are seen in a bed of embryonic, richly vascular C.T. Select an area of active bone formation where the osteoblasts associated with a spicule are large and closely spaced. Observe the intense basophilia of the cytoplasm, due to the presence of large amounts of RNA indicating that protein synthesis is occurring. Mitotic activity is very rare or does not occur in osteoblasts. How are they formed? Sometimes there will be a lighter staining area of bone matrix between the osteoblasts and the deep pink bone spicule. This represents newly formed osteoid which has not yet calcified, and under normal circumstances is an indication of rapidly growing bone. Note the osteocytes within the bone spicules. In spicules covered by thin rather than robust osteoblasts, formation of new bone matrix has slowed down or stopped, and normally no uncalcified osteoid is found in these spicules. Find such spicules covered with thin osteoblasts.

Now, study osteoclasts, which are large, multinucleated cells with slightly basophilic cytoplasm, and are usually several times the size of a robust osteoblast. They may be found on the surface of resorbing bone, sometimes in depressions called Howship's lacunae or they may become separated from the surface and lie in the nearby C.T. (They are more frequently seen on the side opposite that which is undergoing active bone formation.) The osteoclast should be near a bone surface and should have more than one distinct nucleus.

Endochondral Bone Formation

Examine the tibia of a fetal pig, slide #19. This specimen has not yet developed secondary centers of ossification. However, the growth plate at the epiphysis is clearly visible. You will examine the various zone of a growth plate on slide 15. With reversed ocular or scanning objective, locate the two ends of one developing bone and the larger eosinophilic bony trabeculae in the center of the diaphysis. Surrounding the bone is perichondrium (over the cartilage at the ends) and periosteum (over ossified regions). The bony collar forms at the circumference of the developing bone midway between the ends. Observe the trabeculae at the center of the diaphysis which are eosinophilic and contain osteocytes. Their surface is lined by osteoblasts of various sizes. The larger basophilic cells are associated with active deposition of bone. Move toward the end of the bone and notice the presence of pale-staining basophilic structures within the trabeculae. These are remnants of calcified cartilage matrix around which the bone was laid down.

A later stage of endochondral bone formation is shown in a section of femur, slide #15. Starting with the reversed ocular, locate: (1) articular cartilage of the epiphysis, a smooth surfaced region (2) a secondary center of ossification and marrow cavity in the epiphysis between the articular surface and a belt of basophilic cartilage, (3) the epiphyseal growth cartilage, located between the primary and secondary marrow cavities, and (4) the primary marrow cavity in the diaphysis containing eosinophilic bony trabeculae.

Under low magnification locate the epiphyseal cartilage (growth plate). Proceeding from the epiphysis toward the diaphysis, identify and study under higher magnification:

The zone of reserve cartilage, which is nearest to the epiphysis. Note the adjacent bone.
The zone of proliferation. Note the oriented columns of chondrocytes which arise at this stage. Cells in mitosis are present, but difficult to identify. The cartilage is growing longitudinally.
Zone of hypertropy. The cells become larger.
Zone of calcification. The matrix surrounding them calcifies giving a granular appearance, and the cells degenerate.
Zone of ossification. The calcified cartilage is invaded by vascular, osteogenic tissue from the diaphysis. Osteoblasts cover the remnants of calcified (slightly basophilic) cartilage, and form bone (acidophilic) on it.

Joints

Examine the developing joints in slide #19. Observe that the joint capsule extends from one bone to the other, but does not cover the articular cartilage at the apposing bone surfaces. At the inner surface of the joint capsule, a synovial membrane extends into the joint cavity where the contour of the articular cartilage is rounded. Examine the articular cartilage on slide #15 and notice the transition from cartilage to bone of the secondary center of ossification. Observe the smooth surface of the articular cartilage which can be seen on one surface of the bone. Study its structure and note that its surface is smooth and lacks a perichondrium.