lab protocol cartilage - dartmouth.eduanatomy/histo/protocols/lab protocol 2.pdf · lab exercise 2...

Lab exercise 2 – CARTILAGE, BONE AND MUSCLE

Lab protocol

In this lab we will first examine cartilage, bone and muscle at the light microscopic level. We will also consider the process of bone development.

CARTILAGE 1. Slide #51 (frontal section of fetal pig snout; H&E). Find areas of developing hyaline

cartilage (precartilage) in regions surrounding the nasal cavities, especially the nasal septum (see figure below). The young cartilage cells (chondroblasts) have begun to lay down a pale intercellular matrix, thus separating the cells from one another. Between the precartilage and the surrounding loose mesenchyme is a region of condensed mesenchymal cells. This is the future perichondrium. The innermost part of the perichondrium is the chondrogenic layer.

2. Slide #65 [frontal section of the larynx of a rabbit; H&E]. (May include part of thyroid gland). Orient yourself to this section using low power and by referring to the diagram below. Find the areas of mature hyaline cartilage which compose the largest laryngeal plates and the tracheal rings. Identify territorial matrix, interstitial matrix, chondrocytes within lacunae, and note the occurrence of cell nests (aka isogenous nests). Cell nests result from the mitotic division of chondrocytes and are a manifestation of interstitial growth. The different staining of the territorial matrix and the interterritorial matrix has to do with the high concentration of proteoglycans in the territorial matrix as well as the varying thickness and orientation of the collagen fibers between the two zones. Note the absence of blood vessels in the hyaline cartilage.

Chondrocytes are usually shrunken and distorted because a rather long time is required for the fixative to penetrate the dense cartilage matrix.

Look for the perichondrium. Note that the chondrogenic layer is less prominent than in immature hyaline cartilage. Are there areas of calcification within the central regions of some of the cartilages in your specimen? This is a degenerative process.


How do cartilages grow interstitially? How do they grow appositionally?

3. Slide #66 [young rabbit pubic symphysis; H&E]. [See diagram below]. This slide of the pubic symphysis contains hyaline cartilage, fibrocartilage and bone, as well as the surrounding skeletal muscle. Developing bone is present in triangular wedges on each side of the midline cartilaginous symphysis. In most sections the junction of developing bone is marked by a prominent basophilic zone of calcified cartilage. The muscle attaches to the periosteum of the bone and the symphysis by means of tendinous insertions. As indicated in the diagram, the symphysis is a cartilaginous joint (synchondrosis) of limited mobility (amphiarthrosis; same as an intervertebral disc). The center of the joint is mostly hyaline cartilage. There is a transition to fibrocartilage at the inferior and superior margins of the symphysis. This fibrocartilage, in turn, makes a transition to dense fibrous CT (tendon). Fibrocartilage is recognized by the predominance of highly acidophilic type I collagen fibrils in the matrix. This partially overshadows the basophilic staining properties of components of the cartilage matrix ground substance (GAGs, proteoglycans). Hence, in H&E preparations, the matrix of fibrocartilage usually has a faintly eosinophilic or mottled eosinophilic/basophilic appearance. One way that fibrocartilage can be distinguished from dense connective tissue is that the cells (chondrocytes) are contained in lacunae, sometimes with isogenous nests.


4. Slide #64 [intervertebral disc; H&E]. In this slide, the fibrocartilaginous intervertebral

disc is sandwiched between parts of two vertebrae composed of highly eosinophilic bone. Find chondrocytes in lacunae in the fibrocartilage. Note the mottled eosinophilic/basophilic matrix containing coarse bundles of type I collagen fibrils (part of the anulus fibrosus). (In some sections, part of the mucoid nucleus pulposus may be seen). Observe how the collagen fiber bundles of the fibrocartilage blend into the matrix of the surrounding bone creating a strong union between the two tissues. What happens in the common spinal disorder, “ruptured disc”?

5. Slide #56 [section of human external ear (pinna) Verhoeff elastic stain]. Using the

diagram below as a guide. Review the structure of the epidermis and dermis (with associated hair follicles, sebaceous glands and numerous blood vessels). Elastic fibers stain a deep magenta or blackish purple. A segment of elastic cartilage occupies the central part of the section. It has basically the same structure as hyaline cartilage, except the matrix contains an abundance of elastic fibers (magenta staining here). One may be able to find an example of elastic cartilage stained only with H&E in Slide #65 if some of the epiglottal cartilage is present in your particular section. However, with this stain, it is impossible to identify the elastic fibers.

ADULT BONE

6. Slide #71 [mature bone; decalcified, H&E]. Because of the difficulty inherent in

processing bone material (e.g. decalcification, sectioning), your particular section may show artifacts such as tearing of the section or uneven coloration (bone should be eosinophilic with H&E). There also tends to be shrinkage of the marrow away from the bone. Nevertheless, this slide highlights many features typical of mature bone. First examine the cortical bone. There are blood vessels residing in larger holes in the bone (Haversian canals) surrounded by smaller openings (lacunae). Some of the lacunae can be seen to contain osteocytes. Notice that these lacunae are often arranged in a circle around the blood vessel. If you carefully focus up and down you may note that the bone matrix also appears to wrap around the Haversian canals. These lamellae with the


osteocytes that created them comprise the Haversian system or osteon. The outer border of an osteon is marked by a cement line, a region of collagen-poor bone matrix. These cement lines are actually seen to best advantage in those sections that do have some staining artifacts where they are visualized as thin dark lines delimiting an osteon. Cement lines represent weak, fracture-prone areas of the bone. Closing down the iris diaphragm on your microscope, you can better appreciate the lamellar arrangement of the bone matrix as well as some of the details of the canalicular system connecting neighboring osteocytes and the Haversian canal. The canalicular architecture will be seen better in the subsequent slide. Identify some interstitial lamella found amidst the osteons. These lamellae are remnants of former osteons, the other parts of which were resorbed during a previous remodeling cycle. Identify resorption cavities, which are evidence of current remodeling taking place even in this adult bone. You will see these as cavities, much larger than the Haversian canals, within the compact bone. You may also find some scooped out areas (particularly along the endosteal side of the bone) called resorption bays (or Howship’s lacunae). Remember, bone is not a static tissue! The cellular preservation of this specimen is not good enough to allow positive identification of the cells within these cavities. How can you tell which phase of remodeling (resorption, formation) is occurring in a particular cavity? Think about the type of cells you would find during each process. There are lamellae just beneath the periosteum that encase the entire bone. These are the outer circumferential lamellae, while there are also circumferential lamellae just beneath the endosteum, the inner circumferential lamellae (see figure below; not all sections will clearly show both of these layers but one of them should be present). Now look at the marrow cavity and identify the trabecular bone (if any is present in your slide). Being mature bone its organization is also lamellar, though this may be more difficult to appreciate because of the numerous oblique sections resulting from the 3-dimensional, sponge-like arrangement of the trabeculae (try closing down your iris diaphragm to observe the matrix). While occasional concentric arrangements of bone tissue may be found in a trabecula, these are not true osteons. Trabeculae are thin enough to derive their nourishment from surrounding vessels and therefore lack the central blood vessel that characterizes an osteon of mature compact bone.


7. Slide #70 [dried, polished compact diaphyseal bone; unstained]. The piece is unfixed, unstained and not decalcified. It is oriented in the transverse plane. Using the figure above, identify typical concentric-ringed Haversian systems (osteons). Do not attempt to study this slide with your oil immersion lens. The central Haversian canals (which in life contained blood vessels) are filled in with bone dust from the polishing process and appear as dense black circles. (What are Volkmann’s canals? Find one.) Surrounding the canals are concentric rings of compact bone containing lacunae and connecting canaliculi (where osteocytes and their processes once dwelt). What was the nutritional significance of the canaliculi to the osteocytes? Look for interstitial lamellae. Realize that these represent remnants of old Haversian systems.

BONE DEVELOPMENT is a complex process, resulting in the adult appearance of bone. 8. Slide #51 [fetal pig snout; H&E]. Look at this section once again (see figure below)

showing developing trabeculae of bone found around the base of the cranium and in the lower jaw. Numerous osteoblasts are seen to be covering the trabeculae. Surrounding the trabeculae is loose mesenchyme containing many blood vessels. Hematopoietic tissue (marrow) has not yet developed. Give particular attention to the morphology of the osteoblasts, noting their very basophilic cytoplasm (indicative of what?) and large, rounded nuclei. Note that the nuclei of the osteoblasts are generally positioned in the cell away from the side contacting the bone.


9. Slide #67 [human fetal finger; decalcified, H&E]. This is a longitudinal section of a fetal finger (see figure below). Three phalanges will be seen in most sections. Using your lowest power objective, note that both ends of the phalanges (except the distal) are composed of typical hyaline cartilage. The cartilaginous ends are known as the epiphyses. Note that they form the articular surfaces of the phalanges and border joint cavities, which are lined by synovial membranes. The central regions of the phalanges (the shafts or diaphyses) are composed of cylinders of mostly compact bone (staining uniformly eosinophilic). The marrow cavity is enclosed by bone and is here filled with red marrow. This is active hematopoietic (blood forming) tissue which we will study in later lab. The shafts of the phalanges are attached to their epiphyses by a region composed of calcified cartilage (dark purple) and newly formed bone (pink-red). This transition region is known as the metaphysis and is the site of endochondral bone formation (in this case, at a fairly early stage). Study the epiphyseal and metaphyseal regions with low and then high power. Note the enlargement (hypertrophy) of chondrocytes which are arranged in columns in the part of the epiphysis bordering the metaphysis. These are the oldest chondrocytes - the matrix surrounding them has become calcified (hence the deep basophilic staining). As the chondrocytes die, new bone is deposited on the remaining framework of calcified cartilage matrix as osteoblasts remove the calcified cartilage. Note the cores of calcified cartilage in the trabeculae of new bone that are forming in the metaphysis (“mixed trabeculae”).


10. Slide #68 [fetal rabbit tibia; H&E]. Orient the section using your lowest power objective and the diagram below. Identify: epiphysis, metaphysis, marrow cavity, diaphyseal bone, and periosteum. Note that with the staining technique used here, the hyaline cartilage of the epiphyseal hyaline cartilage matrix stains faintly acidophilic; calcified cartilage, dark purple; bone, bright red. Using low and then high power, study the epiphyseal-diaphyseal junction region in more detail. Working from the epiphysis toward the diaphysis, identify:

• Zone of reserve ("resting") cartilage (unmodified hyaline).

• Zone of proliferating cartilage - clusters (cell nests) of chondrocytes undergo successive mitotic divisions to form columns of cells separated by deeply staining matrix.

• Zone of maturing cartilage - cell division ceases, chondrocytes increase in size.

• Zone of hypertrophy and calcification - hypertrophied chondrocytes dying or

lysed away, matrix material calcified, invasion of capillaries and osteogenic cells.

• Metaphyseal (osteogenic) zone - lamellae of new bone deposited on remnants of calcified cartilage.

Observe the osteoblasts covering the lamellae of new bone (some may have been detached during preparation of the section). Additional osteoblasts are seen lining the walls of the marrow cavity of the diaphysis. These constitute the endosteum of the bone. Note the numerous blood vessels in the marrow adjacent to the bone trabeculae. What is the origin of osteoblasts?


Find osteoclasts attached to the bone trabeculae in, just below, and lateral to the metaphyseal region. (Do not confuse these giant, multinucleated cells with another type of giant cell, megakaryocytes, found in the bone marrow. The latter are multilobed but not multinucleated.) What is the function of osteoclasts? What is their origin? Look for early signs of secondary ossification center development in the epiphysis (may not be present in all slides).

11. Slide #69 [fetal long bone; decalcified, H&E]. Referring to the diagram below, first identify the epiphysis, metaphysis, and diaphysis regions of the bone. Note the hyaline cartilage covering the articular (free) surface of the epiphysis. Cartilage will persist here into adulthood. Note: there may be a portion of the attached ligament of the head of the femur. Beneath the articular cartilage is the cancellous bone of the epiphysis that has formed from the “secondary” or “epiphyseal” ossification center. Much of this bone is still immature (woven bone), but some mature (lamellar) bone may be seen. How may these two types of bone be distinguished? Osteoblasts, contributing to well-defined endosteal layers, as well as numerous osteoclasts can be seen in this section. At the margins and near the epiphyseal disk cancellous bone is being converted to denser, compact bone. The histological “snapshots” provided by this slide offer an excellent opportunity to study the development of the earliest Haversian systems (osteons) typical of this type of bone. Look next at the epiphyseal disk. Note its intimate union with the bone of the epiphysis and the now only very narrow zone of reserve cartilage. What is meant by “closure” of


the epiphysis? How would an epiphyseal plate appear on a radiograph? Identify, as in the previous slide, the zones of proliferating, maturing, and calcifying cartilage and their junction with the metaphysis. In the metaphyseal region note again the remnants of calcified cartilage matrix upon which bone is being deposited by osteoblasts. Also identify some of the many osteoclasts. Can you define any resorption bays (Howship’s lacunae)? Understand the process of bone resorption occurring in the resorption bay.

12. Slide #72 [fetal long bone x-s; decalcified, H&E]. This is a cross section of the mid-shaft region of a developing fetal long bone of about the same fetal age as the bone in Slide #69. This slide illustrates the processes of circumferential growth and remodeling of this region of a long bone. You should understand that in the developing long bone the transformation of the cancellous periosteal bone collar into a compact bone shaft begins simultaneously with the initiation of endochondral ossification at the epiphyseal-metaphyseal complex. Survey the section using low and medium magnification. Note the highly irregular periosteal (outer) and endosteal (inner) surfaces of the shaft primary spongiosa (composed of immature woven bone). The periosteum is very distinct and its inner osteogenic and outer fibrogenic zones easily discerned. The latter provides attachment for the tendons of the developing muscles surrounding the shaft. Centrally note the developing marrow cavity and the abundance of active hematopoietic tissue (blood forming cells). Now study the section with higher power. Where do you find the greatest number of bone forming cells (osteoblasts)? Where do you find the greatest number of bone resorbing cells (osteoclasts)? How do your observations help explain shaft modeling?


Where in the section is the process of compaction (primary osteon formation) occurring? What is the significance of this process?

13. Slide #90 [rat spinal column, in situ; decalcified, H&E]. Look for portions of the bony vertebrae and study these at higher power. You should be able to find examples of dense lamellar bone (arch and spine of vertebra) as well as typical cancellous bone (body of vertebra). Since some endochondral bone formation is still occurring in this animal, much of the bone is of the immature type. Observe the abundant red marrow (hematopoietic tissue) surrounding the bony trabeculae in the body of the vertebra. Can you find remnant strands of calcified cartilage? How does the endochondral ossification process in an irregular bone, like the vertebra, differ from that of a typical long bone?

MUSCLE Skeletal Muscle 14. Slide #76 [teased skeletal muscle; H&E]. Individual muscle fibers, which are

individual muscle cells, have been teased apart and mounted without sectioning. Observe that skeletal muscle fibers are cylindrical and do not branch. Note that fibers have multiple, peripherally-located nuclei. With careful analysis, one can discern that the myofibrils are organized into sarcomeres, the portion of a myofibril located between two Z bands. The sarcomere can be subdivided into a darker central A band (which is divided by the M band) and two lighter I bands with the Z line (Z disc) splitting the I bands.

15. Slide #78 [transverse section of an entire small skeletal muscle]. Analyze the

connective tissue sheath system (endomysium, perimysium, epimysium) of skeletal muscle. Fixation shrinkage of the muscle fibers makes it easy to distinguish endomysium and perimysium since the fibers have shrunken away from the connective tissue a little. Only a portion of epimysium may be seen in most slides. Note the many blood vessels running within the endomysium. Skeletal muscle has a high metabolic rate and therefore requires an abundant blood supply. Consider how the connective tissue sheaths support the muscle fibers and harness their contraction.

Look for a neuromuscular spindle on this slide (in the on-line section it is slightly above the middle of the section). It would be cut in cross-section (as are the rest of the muscle fibers). Notice that the intrafusal muscle fibers (the ones inside the spindle) are much smaller than the extrafusal fibers (the rest of the skeletal muscle fibers outside of the


spindle). Because the spindle is connected to connective tissue in parallel with the extrafusal muscle fibers, it detects stretch of the muscle. The fact that intrafusal muscle fibers have contractile properties makes them adjustable.


16. Slide #66 [pubic symphysis] and Slide #78 [tongue]. You must be able to recognize skeletal muscle when sectioned in both the transverse and longitudinal plane, as well as obliquely. To this end, study skeletal muscle fibers in these two slides, which have skeletal muscle cut in several planes. Also, observe the histology of the attachment of muscle to tendon in the pubic symphysis slide. Review the character of attachment of tendon to bone and now summarize the structures involved in transmitting the force of a muscular contraction to bone.

17. Slide #96 [neuromuscular junction, gold chloride stain]. Find and study a motor end

plate in this preparation of teased skeletal muscle fibers (see figure below). A motor unit is all of the muscle fibers innervated by a single motor axon.

Smooth Muscle

18. Slide #79 [teased smooth muscle fibers]. Study some isolated fibers and note the

following: • approximate length and spindle-like shape. • number, position, form, and chromatin of nucleus. • absence of distinct myofibrils or sarcomeres in the cytoplasm.

19. Smooth muscle varies greatly in distinctness and arrangement in different organs and

types of preparations. Its identification is sometimes difficult. Hence, it should be observed in slides from several parts of the body. In the connective tissue stroma of the ectocervix, Slide #54, muscle cells are arranged singly or in small groups. This is also true in the body of the uterus, Slide #80. Note that in H&E stained sections it can be difficult to distinguish smooth muscle cells from fibroblasts with connective tissue. The nuclei of smooth muscle cells are generally more euchromatic than fibroblast nuclei. They also tend to be less angular and more cigar or cucumber shaped. Occasionally they appear crinkly, particularly if the smooth muscle cell is contracted. Smooth muscle fibers are stained red in this trichrome stain.

20. In the intestinal wall, Slide #28, smooth muscle fibers are packed densely together. The smooth muscle cells are arranged in two distinct layers at right angles to each other with intervening connective tissue. The outer layer of smooth muscle is longitudinally directed, while the inner layer is circumferential to the lumen. Locate these layers.


Briefly examine the smooth muscle from other locations (Slides #31 Urinary Bladder & #32 Esophagus) in order to expand your understanding of its variable appearance.

Cardiac Muscle 21. Slide #81 [teased cardiac muscle fibers; H&E]. Observe the striated pattern, location

of the nucleus and branching pattern of teased cardiac muscle fibers.

22. Slide #82 [cardiac muscle, left ventricle; H&E]. Identify some longitudinally oriented cardiac muscle fibers and observe the location of the nuclei. How many are there in each cardiac muscle fiber? Identify intercalated discs. Try to find some branching cardiac muscle fibers (see the figure below). Note that it may cross the fiber at one level or only partially cross the fiber and apparently disappear. What is the structure of the intercalated disc as revealed by the electron microscope (figs. 3-5, “EM of muscle” module of virtual histology site)? Study the cross-striations of the muscle fibers and note that the sarcomere and its subunits are not as clearly delineated as in skeletal muscle.

23. Slide #113 [heart & A-V valve; Masson trichrome]. The cardiac muscle fibers are not

as easy to visualize with this stain. However, you may be able to make out the branching pattern of cardiac muscle fibers, the location of intercalated discs and the fact that cardiac muscle fibers are not all oriented in parallel rows.

lab protocol cartilage - dartmouth.eduanatomy/histo/protocols/lab protocol 2.pdf · lab exercise 2...

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