Histologic:Chapter 13

Skeletal Muscle

Skeletal muscle was discussed in detail above in the section on contractile cells (chapter 5).

Cartilage

Cartilage is an avascular connective tissue specialized to provide support with some flexibility. The ground substance of cartilage is firm and compact. It consists of chondromucoprotein that stains with basic dyes. Of the three types of cartilage, hyaline cartilage is the predominant form; it occurs in more areas than either elastic cartilage or fibrocartilage.

Hyaline Cartilage

Slide 2, Trachea (H&E)

On slide 2, Trachea (H&E), identify the hyaline cartilage which provides support for the softer tracheal tissues. The cartilage enables the trachea to maintain patency. Scan the slide with low power noting the hyaline cartilage.

With medium power (10X) scan the width of the cartilage plate noting that a dense fibrous connective tissue, the perichondrium, lies on both sides (actually surrounds) the cartilage. Chondrocytes lie within lacunae. The cartilage cells are shrunken by fixation and histological processing. Adjacent to the perichondrium, the lacunae are small and flattened, whereas the lacunae deeper within the cartilage plate are larger and rounded. The chondrocytes occupying the flattened lacunae represent young cartilage cells that have differentiated from the cellular perichondrium (not present in this mature cartilage). This represents appositional growth of the cartilage. In growing cartilage, the perichondrium consists of an outer fibrous layer and an inner cellular layer. The chondroblasts of the cellular layer arise by division of their own kind and by the differentiation of mesenchymal cells and fibroblasts into cartilage- forming cells. A cellular layer is not readily apparent when the cartilage is not actively growing.

Note the thin zone of darker basophilic matrix immediately adjacent to many of the rounded lacunae. The basophilic zone represents the capsule or the “lining” of the lacunae. The capsule is not a membrane. It is a basophilic rim of concentrated chondromucoprotein and only a few collagenous fibers. The capsule is the youngest part of the matrix. The less basophilic-staining matrix surrounding the capsule is the territorial matrix. The matrix between cells or groups of cells is the interterritorial matrix. A group of two or more lacunae may lie separate from other similar groups of lacunae. Each group is referred to as an isogenous group. The cells of an isogenous group represent the offspring of a single chondrocyte that underwent mitosis and later the daughter cells produced matrix material between them. In this manner cartilage grows by the interstitial (internal) method. A cell nest refers to two chondrocytes occupying the same lacunae.

With high power (40X) note the shrunken appearance of the chondrocytes. In the living state chondrocytes fill the lacunae. In our preparations, the chondrocytes may appear to have protoplasmic processes that extend to the capsule. The nucleus is quite dark and wrinkled. Only in the best preserved nuclei is a nucleolus seen. The vacuolated appearance of the cytoplasm results from the loss of glycogen and fat during fixation and processing of the tissue.

Slide 33, Lung and Bronchus (H&E)

On slide 33, Lung and Bronchus (H&E), identify hyaline cartilage in the wall of the bronchus. Here there are several cartilage plates rather than one C-shaped plate.

Elastic Cartilage

Slide 7, External Ear (Verhoeff)

On slide 7, External ear (Verhoeff), identify the elastic cartilage. Verhoeff’s stain demonstrates the elastic fibers by staining them deep purple or blue-black. Note how the fibers branch, interlace and run in all directions. They diverge around the lacunae forming a network that provides the cartilage with flexibility. Although not seen in the preparation, collagenous fibers occur among the elastic fibers. The nuclei of the chondrocytes are well preserved. The cytoplasm of the cells is not stained well. Flattened lacunae occur at the periphery of the cartilage. Cell nests and isogenous groups can be identified. Observe the limits of the perichondrium.

Slide 84, External Auditory Meatus (Verhoeff)

On slide 84, External auditory meatus (Verhoeff), identify the elastic cartilage in the wall of the external auditory tube. On this section, note that the feltwork of elastic fibers is loose and blends with the perichondrium. In this preparation, it is quite evident that the elastic fibers are more closely packed towards the center of the cartilage than at the periphery. This is a general characteristic of elastic cartilage. Note also the elastic fibers in the dense irregular connective tissue, which occupies areas between other structures.

Slide 111, Epiglottis (H&E)

Identify the elastic cartilage as seen with routine H&E stain. Elastic fibers stand out clearly, densely packed in the interior of the plate, thinner and less dense at the periphery. Chondrocytes are shrunken, and many have dropped out of their lacunae.

Fibrocartilage

Slide 8, Intervertebral Disc (H&E)

On slide 8, Intervertebral disc (H&E), locate the spongy bone of the vertebrae and the cartilage which unites the two bones. This cartilage consists of both the hyaline and fibrocartilage types. Fibrocartilage forms the middle portion of the disc with the articular hyaline cartilage at the periphery adjacent to the bone. The fibrocartilage stains pink in the preparation, whereas the hyaline cartilage is almost purple. Characteristic fibrocartilage is best studied at the lower border of the disc lying just outside the disc proper. The fibrocartilage in this position associates with the spinal ligaments and with tendons. Note the properties of the fibrocartilage. The chondrocytes may either lie in rows between heavy bundles of collagenous fibers or be randomly distributed. The lacunae are considerably smaller than is hyaline or elastic cartilage. Typical hyaline cartilage matrix is seen only immediately adjacent to the lacunae. It is important to remember that fibrocartilage always blends into either hyaline cartilage and/or a dense connective tissue. Also, fibrocartilage lacks a perichondrium.

Bone

Bone is a hard connective tissue in which the fibers are impregnated with mineral substance, chiefly calcium phosphate and calcium carbonate. Bone is classified according to its morphological appearance as either compact or spongy (cancellous) bone. The only difference in the two kinds of bone is in the size and number of spaces present. Compact bone is more solid than spongy bone, the latter being a latticework of slender, irregular trabeculae. Bone is also classified according to its origin. Membrane bone (intramembranous bone) develops within a connective tissue membrane (“a mature mesenchyme”) whereas endochondral bone (intracartilaginous bone) develops by replacing a cartilage template that is formed before bone replaces it.

Slide 9, Ground Bone (dried)

On slide 9, ground bone (dried), may be seen the main features of compact bone in which the organic material is missing. In such a preparation, the organic components are lost when the bone is dried. Then the remaining bony material (inorganic) is ground thin enough to transmit light for microscopic study. The overall architectural features of the bone persist.

Note the following:

• Osteons (= Haversian systems) consist of central canals, concentric lamellae, and lacunae. In this slide they are seen mainly in cross or oblique sections.

• The central canal is 22 to110 μm in diameter. It runs longitudinally in the bone and serves as a channel for blood vessels. It is lined by endosteum (not present in this dried bone).

• Concentric lamellae are successive layers of bone which surround the Haversian canal. They are 3 to 7 μm thick and 4 to 20 in number. By lowering the condenser and decreasing the intensity of light, it may be possible to detect that the lamellar pattern appears to alternate between punctate (a stippled appearance) and striate (a linear appearance) according to the orientation of the collagen fibers in the living state. The collagen fibers alternate in direction in adjacent lamellae to give a striate and punctate appearance depending on whether fibers are cut longitudinally or transversely.

Within or between the concentric lamellae of a single osteon are seen the lacunae connected to each other and to the central canal by the canaliculi. Note: the number of “concentric-appearing layers” of lacunae does not necessarily duplicate the number of lamellae for the osteon since the lacunae may occupy any position with respect to the lamellae. Lacunae can lie within or between lamellae. In the living state, osteocytes occupy the lacunae. Here the lacunae appear dark because of air trapped within them. What is the function of the canaliculi?

Again one may be able to observe a refractile line that seems to limit the boundary of each osteon. This is the cement line.

In addition to the concentric lamellae, interstitial lamellae and circumferential lamellae occur in compact bone. Interstitial lamellae are irregular areas of compact bone occupying spaces between adjacent Haversian systems. They most likely represent the remnants or fragments of Haversian systems that have remained from the microscopic reorganization of earlier generations of Haversian systems in the bone. Remember that bone is a very dynamic tissue and the Haversian systems are constantly being “reworked.” There is constant internal bone destruction and reconstruction so that successive generations of Haversian systems occur. Circumferential lamellae occur at the external and internal surface of compact bone similar to the “layers of plaster used to finish a rough wall.” These lamellae run parallel with the surface, and they are circumferentially oriented to the axis of the bone.

Identify the central or Haversian canals in the center of the osteon. Look for cross connections from one central canal to another in a different osteon; these run obliquely but continue to be surrounded by concentric lamellae.

Look for perforating canals (Volkmann’s canals). They are transverse channels which unite the periosteal surface or the marrow cavity with the nearest Haversian canals (may not be present). Perforating canals appear to pass through the circumferential lamellae, and they seem to orient perpendicularly or obliquely to the lamellae.

Slide 8, Intervertebral Disc (H&E)

On slide 8, Intervertebral disc (H&E), observe the spongy bone with its red bone marrow containing developing erythrocytes and granulocytes, fat cells and megakaryocytes. Note again the hyaline cartilage and fibrocartilage. Here, the bone is in the form of interconnecting beams or trabeculae with large marrow spaces between them. The endosteum lines the bone adjacent to the marrow spaces. This lining is difficult to identify since it consists mainly of condensed reticular connective tissue; it is very thin. Some flattened cells, widely spaced, reside in this layer. They have osteogenic potencies. Since this bone is not actively growing, one does not expect to see a large number of active osteoblasts. In a decalcified preparation such as this, the collagen fibers swell, making it difficult to identify the successive lamellae and the canaliculi. Osteocytes are seen occupying the lacunae.

Joints

Bones are connected to each other by joints, which permit varying degrees of movement between the joined bones. Joints can be divided into two main groups: those that permit limited movement and those that permit free movement. In joints that permit only limited movement, the bones are connected by flexible fibrocollagenous or cartilaginous tissue. Such joints generally occur between the flat bones of the skull or the ribs and sternum.

A clinically important group of joints of limited movement are those between vertebrae.

Slide 8, Intervertebral Disc (H&E)

Slide 8 (H&E) is a section of intervertebral disc. The bodies of the vertebrae are joined to each other by the intervertebral disks to form a long uninterrupted column (vertebral column). The disks are thick rubbery pads which not only act as shock-absorbers but also permit some movement, so that the column is flexible within limits.

Intervertebral disks are composed essentially of fibrocollagenous tissue containing some chondrocytes and cartilage matrix (fibrocartilage, see page 100). The two surfaces in contact with the vertebral bodies each consist of a thin layer of hyaline cartilage covering a concentrically lamellated rubbery structure of fibrocartilage (the annulus fibrosus). At the center of the disk is a soft, semifluid core of soft gelatinous matrix (the nuclear pulposus) which is not seen in this slide.

Joints that allow free movement between adjacent bones are termed synovial joints.

Slide 282 Finger Joint (H&E)

The bone ends are held together by bands of fibrocollagenous tissue (ligaments), which may be external or internal to the joint cavity; external ligaments surround a fibrous capsule enclosing the heads of the bones, which are separated from each other by a lubricant fluid, synovial fluid. Because the bone ends move against each other they are coated with a smooth, friction-free layer of hyaline cartilage (articular cartilage), and the synovial fluid provides a thin lubricant film between the opposing articular cartilages.

The internal lining of the joint capsule is a specialized secretory epithelium, the synovium, which produces synovial fluid.

The synovium is composed of one to four layers of cells which merge on their deep surface with a zone of loosely arranged fibrocollagenous tissue containing adipocytes, fibroblasts, mast cells and macrophages. This deep layer merges with the denser fibrocollagenous tissue of the joint capsule.

Synovial cells vary from flat, mesothelial-like cells through to spindle-shaped, polyhedral or cuboidal cells.

Ligaments, composed of dense collagenous tissue, stabilize joints.

Structures around the joint that prevent excess movement include collagenous external ligaments and tendinous muscle attachments (see slide 56, Tendon, H&E). External ligaments surround articular (synovial) joints, attaching one bone to the other over the outer surface of the joint capsule.

Ligaments are composed of tightly packed collagen fibers all running in the same direction, with compressed fibrocytes in between; thus they resemble tendon, but differ from tendon in that they contain elastic fibers. Ligaments strengthen the joint; they permit normal movement but prevent overflexing or overextension. The attachment of ligament to bone is similar to that of tendons.

In some complex joints there are internal ligaments (e.g. cruciate ligaments in the knee joints) to prevent overstretching or twisting, and fibrocartilaginous menisci (also in the knee) to stabilize and guide gliding movements.

Slide 283, Knee Joint (rat) (H&E)

Slide 283 (H&E) is a knee joint of a rat. Note bone, articular cartilage, tendons and ligaments.