Prelab Exercise 2. Prelab Exercise 2 CARTILAGE, BONE & MUSCLE

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1 Prelab Exercise 2 CARTILAGE, BONE and MUSCLE CARTILAGE Cartilage is a specialized connective tissue in which the extracellular matrix (fibers and ground substance) is modified to provide a relatively rigid support for certain parts of the body as well as a smooth, slippery surface for articulation of some joints. Most of the fetal skeleton is composed of cartilage. The bulk of this is replaced postnatally by bone. (In what sites in the adult body does cartilage persist?) The objective of your microscope study is to observe the modifications, which occur in each of the three basic components of connective tissue to create a type of cartilage, which is adapted to the mechanical needs of specific areas. The three types of cartilage found in the adult (hyaline, fibro-, and elastic) are distinguished primarily on the basis of the visibility, nature, and arrangement of the fibrous component of the matrix. Hyaline Cartilage Developmentally, young cartilage cells (chondroblasts) differentiate from mesenchymal cells and begin 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. Mature hyalin cartilage consists of matrix and chondrocytes within lacunae. Note that several chondocytes may exist in one lacuna (isogenous nest). These cell nests result from the mitotic division of chondrocytes and are a manifestation of interstitial growth. The cartilage matrix immediately surrounding the chondrocytes is deeply basophilic. This constitutes the territorial matrix (capsular matrix), as distinguished from the more lightly stained interterritorial matrix found more distant from the cells. 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. The perichondrium borders the cartilage, with the part closest to the cartilage being the chondrogenic layer (less prominent than in immature hyaline cartilage). This is the area of appositional growth. There are no capillaries in the matrix of cartilage (it is avascular). Thus, chondrocytes must be nourished by slow diffusion from blood vessels located in the fibrous layer of the perichondrium. Cartilage has a low rate of metabolic activity and, regeneration may be very slow or absent following injury. There may be areas of calcification within the central regions of cartilage. This is a degenerative process. Cartilage, unlike bone, can enlarge by interstitial growth as well as appositional growth. Where do these two growth processes occur? Fibrocartilage Fibrocartilage is found most commonly as an intermediate or transitional tissue between dense regular CT (tendons, ligaments, aponeuroses) and hyaline cartilage or bone. Hence, a distinct perichondrial layer is usually absent. Most of the fibrocartilage of the human body is located in the intervertebral discs, which constitute about one-fifth of the vertebral column height in the young adult. (In what other specific sites of the body is fibrocartilage found)? Fibrocartilage is the strongest of the cartilage types and is best adapted for withstanding mechanical stress (tension). However, as with all cartilage types, its capacity for regeneration after injury is very limited in the adult. Like all cartilage types fibrocartilage is characterized by the presence of native cartilage cells (chondrocytes) within lacunae in the matrix, although in the case of fibrocartilage these cells tend to be 1

2 fewer in number and more widely dispersed. (They are rarely found in isogenous cell clusters as in hyaline cartilage). Fibrocartilage is also unique in that its matrix contains a greater abundance of collagen fibrils, often organized in distinct bundles oriented along lines of stress. Unlike hyaline cartilage these collagen fibrils are mostly type I rather than type II. Elastic Cartilage BONE As the name implies, elastic cartilage is more elastic than regular hyaline cartilage. This is useful where some rigidity, but also flexibility of cartilage is important. Classic examples of this are the external ear and the epiglotis of the larynx. This cartilage type contains many elastic fibers, in addition to type II collagen fibers. It is really only distinguishable if stained for the elastic fibers. The matrix of bone is specialized to provide a means of rigid support. This is accomplished by the deposition of inorganic salts in an organic matrix. Bone is formed either directly in young fibroelastic connective tissue (intramembranous) or by the replacement of hyaline cartilage (endochondral). Some bones (e.g., those of the calvaria) are formed only by the first method, whereas in the development of others (e.g., long bones) both processes are involved. Keep in mind the great practical importance of bone histogenesis. In every healing fracture you deal with in the future, the formation of callus and the reunion of the broken surfaces involve recapitulation of intramembranous bone formation. Also, damage to cartilage can trigger a transformation to bone, much as is seen developmentally. Adult Bone Long bones consist of a diaphysis (shaft), a metaphysis (the expanding part toward the end) and an epiphysis (the larger end of the bone, often covered by articular cartilage). The core of the bone usually consists of a marrow space. There are two basic types of mature bone: compact (cortical) and cancellous (spongy). Compact bone makes up most of the shaft of long bones, while cancellous bone is usually in a marrow space and surrounded by the bone marrow. Mature bone takes on a layered configuration, called lamellar bone. These layers are laid down by ostoblasts that exist on the outer and inner bone surfaces (periosteum or endosteum, respectively), or that arrange themselves circumferentially around blood vessels 2

3 within the bone (as Haversian systems or osteons). As osteoblasts become surrounded by bone, they change their name to osteocytes. The figure above shows an osteon (on the right), with lacunae (containing osteocytes) surrounding it. These lacunae connect to each other so that the osteocytes in one lacuna can contact those around it. This is how nutrients are passed from the blood vessel to the osteocytes. The blood vessels in the Haversian canals are interconnected by transversely running Volkman s canals. The outer border of an osteon is marked by a cement line, a region of collagen-poor bone matrix. Interstitial lamellae are found between osteons. These lamellae are remnants of former osteons, the other parts of which were resorbed during a previous remodeling cycle. While the layers of bone (lamellae) of an osteon surround the blood vessel, the layers of bone that are laid down by the periosteum and endosteum wrap all the way around the bone, on the inside or outside of the cortex of the bone. These are called the inner or outer circumferential lamellae. The trabeculae of cancellous bone don t, as a rule, have osteons. Instead, they consist of lamellae of bone in parallel with the surface of the trabecula. Mature, lamellar bone is not static. Instead, it is constantly being remodeled. There are resorption cavities, tunnels in the bone that are carved out by burrowing osteoclasts. These are highly eosinophilic, multinucleated cells that break down bone. There are also resorption bays (Howship lacunae) that scoop out divots, mostly on the endosteal surface or on the trabecule. This bone must be replaced by new bone in order to avoid weakening the bone. The strength of bone is basically a balance between bone production and resorption. Bone Development Bone development is a complex process that, to one extent or another, continues throughout life. We will focus on early development, which either begins with ossification of a hyaline cartilage model (endochondral) or of a connective tissue membrane model of the bone (intramembranous). In the adult bone, one could consider the ossification that occurs at the endosteum and periosteum to be a form of intramembranous ossification. Ossification usually occurs in the diaphysis first. A secondary ossification center then develops in the epiphysis, leaving a cartilage plate in between. This is the epiphyseal plate and, because it is hyaline cartilage, this can grow by interstitial growth. This is the principal way that bone lengthens. In the region of the epiphysial plate there are stages of ossification. 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 osteogenic cells. Metaphyseal (osteogenic) zone - lamellae of new bone deposited on remnants of calcified cartilage. Osteoblasts will cover lamellae of new bone, contributing to new layers of bone and becoming osteocytes. Additional osteoblasts are seen lining the walls of the marrow cavity as endosteum of the bone. Note the numerous blood vessels in the marrow adjacent to the bone trabeculae. Find osteoclasts are common in developing bone. Not only do they dissolve the calcified cartilage, but they also break down bone, so that it can be remodeled. These cells are multinucleated and eosinophilic. Not 3

4 only do osteoclasts scoop out bone from the surface (resorption bays or Howship lacunae) but they also tunnel into the bone, allowing creation of new osteons. The alteration of the size and shape of bones by bone formation and resorption at different surfaces and rates during the growth process is known as remodeling. Remodeling serves primarily to alter the amount of bone that is present and to determine the final form of the bone. During normal growth of a long bone the rates of external apposition of new bone and internal resorption are coordinated so that the cylindrical shaft expands markedly in diameter while the thickness of its wall and the marrow cavity increase slowly. CHECK LIST Understand the developmental processes involved in cartilage and bone and the relationship between this two specialized types of connective tissue. CARTILAGE: Know the three types of cartilage based on general morphology, location and cellular and fibrous structure. Identify and define: -hyaline cartilage (type II collagen) -fibrocartilage (type I collagen present) -elastic cartilage (elastic fibers present) -chondroblasts -perichondrium/chondrogenic layer -matrix, (territorial and interterritorial) -chondrocytes -lacunae -isogenous cell nests/interstitial growth -metachromasia of territorial matrix/interterritorial matrix -calcified cartilage -tendon -EM of chondrocytes and cartilaginous matrix BONE: Know the morphology of bone development as well as gross parts of a long bone. Identify and define: -trabecular = spongy = cancellous bone - compact = cortical bone -intramembranous ossification - very important to understand all steps -appositional growth -epiphysis, metaphysic, diaphysis -marrow cavity -endochondral bone formation - very important to understand all steps -epiphysial plate/disk/line -5 zones of maturation within the epiphysial disk -immature (woven) bone -mature (lamellar) bone -osteoblasts -periosteum -endosteum -osteocytes -osteoclasts -osteoprogenitor cells -osteoid -osteons (Haversian system/canal) -lamellae: inner/outer circumferential lamellae; interstitial lamellae -canaliculi -resorption cavities -Volkmann s canal 4

5 MUSCLE TISSUE The structure of muscle cells (fibers) reflects their specialized capacity for contraction. The cells are much longer than they are wide and are usually arranged in parallel sheets or bundles. Contraction invariably involves shortening of the long axes of the cells. Common to all muscle cells are cytoplasmic myofilaments, actin and myosin, which are involved in the dynamics of contraction. Different types of muscle cells vary markedly in form, size, structure, innervation, and the force with which they contract. Considerable variation occurs in muscle cells of a given type depending on their location and function. SKELETAL MUSCLE (striated, voluntary) Morphology. Individual skeletal muscle fibers (i.e., muscle cells) are cylindrical and do not branch. They are multinucleated, with peripheral nuclei. The striations are due to the arrangement of myofibrils. The sarcomere (the portion of a myofibril located between two Z bands) is the unit of organization. Each sarcomere can be subdivided into a darker central A band (which is divided by the M band) and two lighter I bands. Compare the appearance of skeletal muscle as seen with the light microscope with that seen with the electron microscope (refer to illustrations in your textbooks and figs. 6-9 of the EM of muscle from the virtual histology site). What elements comprise the bands within the sarcomere? The transverse tubule (T-tubule)-system passes through the muscle fiber at the level of the junction of the A and I bands. It is bracketed by two tubular elements of the sarcoplasmic reticulum. This mechanism is important for extending membrane excitation into the center of the muscle fiber and for release of stored calcium from the sarcoplasmic reticulum. This, in turn, is the necessary element in excitation/contraction coupling. Connective tissue components. The connective tissue sheath system of skeletal muscle consists of endomysium, perimysium, and epimysium). It is via these connective tissue investments that blood vessels get to the Muscle-tendon attachments. The connective tissue of the muscle blends with tendon at the musculotendinous junction. Additionally, there are interdigitations of the collagenous fibers with the end of muscle fibers with apparent anchor points between actin inside the muscle fibers with specializations that attach to collagen extracellularly. Motor nerve terminals/sensory nerve receptors in skeletal muscle. Each skeletal muscle fiber has one neuromuscular junction (motor end plate). However, a single motor nerve fibers innervates many muscle fibers (this collection of muscle fibers innervated by one motor nerve fiber is called a motor unit. 5

6 Muscle proprioception. Neuromuscular spindles and Golgi tendon organs are highly specialized sensory receptors that are a part of the proprioceptive component of the somatosensory system. Muscle spindles are comprised of a bundle of modified skeletal muscle fibers ( intrafusal fibers) that are organized in parallel with the major muscle fibers of the muscle ("extrafusal"muscle fibers). The intrafusal fibers have a motor innervation (gamma motor nerve fibers) and a sensory innervation (the most important is the annulospiral ending). This sensory receptor axon detects stretch of the muscle. The sensory nerve fiber relays this information to the spinal cord, where reflexes of varying complexity are activated to maintain posture or to regulate the activity of opposing muscle groups involved in motor activities such as walking. Neurotendinous spindles (Golgi tendon organs) are located near the junction of tendons with muscles. They provide the CNS with information concerning tension within a tendon. In concert with the muscle spindle, they provide information that allows for regulation of muscle tone. One of these structures, the muscle spindles, may be found in your slides of skeletal muscle tissue. SMOOTH MUSCLE (non-striated, involuntary) Smooth muscle fibers are spindle shaped and may appear as sheets, or as individuals scattered in connective tissue. They do not have sarcomers, even though they do have actin and myosin. They contain a single nucleus that is more euchromatic than most fibroblasts. The nucleus is cigar to cucumber shaped and occasionally takes on a corkscrew appearance when the muscle is contracted. Smooth muscle cells may be arranged singly or in small groups ( multiunit organization ). They can also be packed densely in organs such as the GI tract ( visceral/unitary organization ). In that location they appear in distinct layers at right angles to each other, with intervening connective tissue. Examine the ultrastructure of smooth muscle cells in figure 1 of the EM of Muscle module on the virtual histology site. CARDIAC MUSCLE (striated, involuntary) see the figure to the right Cardiac muscle is found only in the wall of the heart (the myocardium). These fibers have a single nucleus and appear in branched chains. Individual cells are connected at intercalated discs, which have an irregular organization (they don t appear in a straight line). This not only anchors one cardiac muscle cell to the next but also facilitates the spread of excitation. The structure of the cardiac sarcomere is similar to skeletal muscle fibers except that there is a dyad (instead of a triad, as in skeletal Cardiac Muscle 1. Central nucleus; 2. Branching; 3. Intercalated Discs; 4. Striations of muscle fibrils 6

7 muscle), with one T tubule in relation to one sarcoplasmic reticulum element. Also, this dyad is located at the Z line. 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. CHECK LIST Identify the three types of muscle in light microscope and electron microscope images. -skeletal (voluntary, striated) -smooth (involuntary, non-striated) -cardiac (involuntary, striated) Identify the connective tissue sheath system of skeletal muscle -epimysium -perimysium -endomysium Be able to define and to identify the following terms: -myofibrils -sarcoplasm -sarcolemma -sarcoplasmic reticulum -sarcomere -transverse tubule system -neuromuscular junction -motor unit -muscle proprioception including muscle spindles -neurotendinous spindles (Golgi tendon organs) -intercalated disc Understand the fibrillar and banding patterns of skeletal and cardiac muscle shown in both light and electron microscopic images. Identify I, M, A, Z, H bands. Know the location of the contractile and regulatory proteins associated with each band. Recognize the diversity, arrangement and location of smooth muscle. 7