Cartilage Lab

Cartilage and Connective Tissue Fibers
Laboratory 5

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Required Reading: Ross and Romrell: Chapter 7, pp. 132-149 and Chapter 5, pp. 94-106

Gartner and Hiatt: Chapter 3, pp. 44-63 and
Chapter 4 (cartilage areas)

YOUR JOB DURING THIS LAB: After completing this assignment you should be able to (a) recognize the different types of cartilage under the light microscope and indicate the cell types present; (b) describe the areas where you might expect to find the different forms of cartilage and why; (c) identify areas containing the major forms of collagen, particularly types I, II, III, and IV and (d) identify the major types of connective tissue fibers and understand their molecular composition. You should also be able to identify dense regular CT, dense irregular CT, loose CT and mucous CT at the level of the light microscope.

CARTILAGE IN ITS VARIOUS FORMS

Hyaline Cartilage

Images to introduce hyaline cartilage

D1: This is a frontal section of the larynx and associated muscles at 2.3X. It shows the relative positions of the cricoid cartilage (a, hyaline cartilage), the infraglottic cavity (b), a thyroarytenoid muscle in a true vocal cord (c), the thyroid cartilage (d, hyaline cartilage), and a laryngeal ventricle (e) which separates a true vocal cord (c) below from a false vocal cord above (tissue near h). Also, sero-mucous mixed glands are shown at (f), the epiglottis (g, elastic cartilage), and a process of the epiglottis (h) in the false vocal cord. TODAY, ONLY WORRY ABOUT THE CARTILAGE COMPONENTS ON THIS SLIDE.

D2: This is an H & E preparation of hyaline cartilage from the trachea. Note the relative positioning of the chondrocytes. Look at the perichondrium. This is a good example of the general appearance of hyaline cartilage.

D3: A higher magnification view of chondrocytes in hyaline cartilage from the trachea. The arrow points to connective tissue at the edge of this cartilage mass. Note the differences in size and shape of the chondrocytes. Which of these are the youngest? How is this region of cartilage growing? Where here would you expect to find chondroblasts, rather than chondrocytes?

D4: A longitudinal view of hyaline cartilage, epithelium, and submucosal glands in the trachea. Which is which? How do you know? Why do you think the cartilage is in the trachea in the first place? Where else can you expect to find it?

Glass slides to view hyaline cartilage.

Slide #93 - trachea: Very similar to image D4, this shows hyaline cartilage to very good advantage. You should identify the perichondrium (which may in some places be pulled away), the cells and lacunae, and the extracellular matrix material. Do you think this cartilage was actively growing when the sample was prepared? On what basis did you decide?

Slide #90 - nasal cavity: This is a good slide on which to conduct a mini-review for yourself. The hyaline cartilage should be obvious as masses of blue, translucent material. Also, locate the epithelium and classify it. Look at the surrounding connective tissue and classify it. Find the glandular ducts; what could they be doing? What do you think the dark pink masses represent?

Hyaline cartilage on this slide is predominantly young, as evidenced by usually only one chondrocyte per lacuna and the presence of a chondrogenic layer (visible sometimes in oblique section with large euchromatic nuclei).

Slide #23 - developing bone/extremities: USE LOW MAGNIFICATION FOR THIS SLIDE in order to orient yourself. Find the hyaline cartilage "model" upon which bone development is occurring (endochondral ossification). On some slides, both the outer fibrous layer of the perichondrium with flattened fibroblasts and the inner chondrogenic layer with chondroblasts having ovoid, euchromatic nuclei will be visible.

Elastic Cartilage

Images to introduce elastic cartilage.

D5: This image shows the region of the false vocal cords at 20X magnification. Elastic cartilage is seen at (a), a part of the epiglottis. Sero-mucous (mixed) glands are indicated at (b) and the epithelium is at (c). (What type of epithelium is it, and why?)

D6: This is another view of the epiglottis showing elastic cartilage (a) and adjacent glands (b).

Glass slides on which to find elastic cartilage.

Slide #56 - external ear: Locate at low power the blue stained regions of elastic cartilage. Note the occasional vertical deeply stained regions. What do you think they are; are they "real"? (NO, they are simply folds that occurred when the tissue was placed on the slide - practice will allow you to distinguish biological information/structure from preparation artifacts.) Observe the change in shape and size of the chondrocytes as they become progressively enclosed by matrix. How does this cartilage compare with the hyaline cartilage examined previously? Why are the elastin fibers hard to visualize here?

Slide #17 - elastic cartilage: This slide of the epiglottis has been selectively stained in order to visualize the elastin fibers. Orient yourself at low magnification and look at the elastic cartilage in the center of the tissue. This is also a good slide to look at other structures - find the elastin fibers in the surrounding connective tissue, for example.

Slide #91/91A - vocal folds/epiglottis: Use the orientation diagrams included here to find the epiglottis. Compare the areas of hyaline cartilage with those of elastic cartilage. Examine the cartilage of the epiglottis at high power and try to locate the poorly stained elastin fibers.

Fibro-Cartilage

Image of fibrocartilage.

D7: Here the fibrocartilage occupies most of the slide. The arrow indicates some of the flattened chondrocyte nuclei. The cartilage matrix stains pink (eosinophilic) because of its collagen fiber content.

Glass slide for fibrocartilage.

Slide #18/18A - pubic symphysis: Fibrocartilage is found at transition areas between hyaline cartilage and dense regular CT. Most of the cartilage here is hyaline cartilage, with only small regions of fibrocartilage located on each side, as the hyaline cartilage merges into the surrounding CT. Note the eosinophilic staining of the matrix caused by the large number of collagen fibers and the absence of any perichondrium. If you have slide #18A, compare the appearance and organization of the fibrochondrocytes there with those shown in your text Plate 15 (Ross & Romrell).

TYPES OF CONNECTIVE TISSUE AND FIBRILLAR PROTEINS

Dense Regular Connective Tissue

This form of CT is characterized by relatively few cells, embedded in extensive amounts of extracellular material, most of which will be regularly arranged collagen fibers. Dense CT is found wherever unidirectional tension must be supported; for example, in tendons connecting muscle and bone, in aponeuroses where specialized tendons connect muscle and bone, and in ligaments.

Images to introduce regular dense connective tissue.

C1: This depicts a tendon cut in longitudinal section. Almost all of the pink material is collagen fibers, very tightly packed in the longitudinal direction and cemented together. The nuclei of fibroblasts (arrows) are elongated because of the dense regular packing of the surrounding extracellular fibers. You cannot distinguish the cell cytoplasm.

C2: Here you see a muscle-tendon junction at 100X magnification. Again, the regularly dense packed collagen fibers are evident, with the interspersed fibroblast nuclei. The "wavy" nature of this tissue is often seen, but is also seen with some samples of both smooth muscle and, particularly, nerve tissue. Learning to distinguish samples such as this from muscle and nerve will take practice.

Glass slides for observation of dense regular CT.

Slide #11 - aponeuroses: here the collagen bundles and fibroblasts are arranged in multiple layers or sheets. Within each layer the fibers follow a parallel wavy course, with the exact direction of the fibers slightly different in different layers. Collagen fibers often go from one layer into an adjacent layer. THE DENSE REGULAR CT IS ORGANIZED IN A REASONABLY WELL DEFINED BAND - THE REST OF THE UNDERLYING CT ON THIS SLIDE IS DENSE IRREGULAR CT.

Slide #13 - skeletal muscle and tendon: Here the muscle is stained much darker than the tendon; today look at the tendon region of the slide.

Dense Irregular Connective Tissue

This tissue is characterized by relatively few cells embedded in collagen fibers, with the fibers not arranged as uniformly as noted in the previous slides. Dense irregular connective tissue is very common, much more so than is dense regular CT.

Image for dense irregular CT.

C3: Compare the increased disorder of the collagen fibers with that seen earlier. The arrowheads point to individual mast cells; these should be seen to better advantage in later slides. Most of the nuclei present here are those of fibroblasts.

Glass slide for dense irregular CT.

Slide #59 - thin skin: A thick band of loose fibrous CT is located beneath the stratified squamous keratinized epithelium. Note the paucity of nuclei in relation to collagen fiber bundles. Much dense irregular CT is also seen on this slide.

Fibers of the Extracellular Matrix

Remember that there are three types of fibers seen histologically; collagen fibers, elastin fibers and reticular fibers. Reticular fibers are similar in biochemical composition to collagen fibers, but will stain differently when placed in alkaline silver salts.

Images for CT fibers

C4: This is an electron micrograph of collagen Type I fibrils in the rat oviductal wall. Note the characteristic periodicity of the fibrils. Note that this whole collection of fibrils forms a single type I collagen fiber as detected at the level of the light microscope.

C5 and C6: These images illustrate the wall of the aorta, a large elastic artery (C5) and a large muscular artery (C6). Here, the wall has many elastin fibers to allow for expansion of the vessel upon acceptance of blood from the heart, and controlled return of the aorta to its unexpanded caliber; this helps maintain adequate blood flow to the body between heart pumps. The elastin fibers are visualized with special stains; normally they appear glassy and translucent (almost invisible).

C11, C12 and C13: These images depict reticular fibers stained with silver salts. Reticular fibers are equivalent to type III collagen and are less than 0.5 um in diameter. They provide a "skeletal" support for other tissues, particularly those containing many lymphoid cells. C11 shows stained reticular fibers in a lymph node and C12 is a higher magnification of a similar field. Note how the reticular fibers surround the lymphoid cells, which are themselves not interconnected by junctions. Image C13 shows the more regularly arranged reticular fibers surrounding the venous sinuses (spaces) in the spleen. the reason for this arrangement should become apparent later in the course when we discuss the structure and function of the spleen in detail.

Glass slides for CT fiber observation.

Elastin Fibers

Slide #9 - mesentery: This is not a section, but rather a whole mount of the mesentery, a thin membranous area of loose connective tissue. The elastin fibers, here unstained by the routine H & E preparation, can be seen as faint glassy fibers forming a network throughout the tissue.

Slide #80 - aorta: Here, in contrast, the elastin fibers can be seen even with the naked eye (although not resolved) as a thick purple/black band.

Collagen Fibers

Slide #11 and Slide #59: You have viewed these already, but they are good examples of the appearance of collagen fibers. Almost any slide that you examine in your collection will exhibit some collagen fibers - try one or two at random and look.

Reticular Fibers

Slide #14 - reticular tissue: This shows the reticular fibers in lymphoid tissue, stained selectively. Compare this slide with:

Slide #61 - lymph node: Here in an H & E preparation the reticular fibers are not very noticeable.

Mucous Connective Tissue

This type of CT has fibroblasts embedded in a viscous ground substance. The appearance of this type of tissue alters as the tissue ages.

Image for mucous CT.

C7: This tissue is taken from an umbilical cord at mid-pregnancy. A few darkly stained nuclei are scattered between the collagen fiber bundles (arrowhead). Most of the space, now appearing open and clear, would normally be fluid-filled.

C8: This slide emphasizes the fibroblasts seen in mucous connective tissue (umbilical cord at 100X).

Glass slides for mucoid CT.

Slide #12 - umbilical cord: This slide shows a large blood vessel, look at low magnification for the tell-tale red blood cells in the lumen, surrounded by mucoid CT. For now, concentrate on the structure of the latter tissue type.

Slide #195 - placenta: Here the CT is mesenchymal, having more mesenchymal cells per unit area than fibroblasts present in the true mucoid CT. The ground substance here is also less viscous than seen in mucoid CT; therefore, it stains poorly.

Loose Connective Tissue

Loose CT is characterized by a much more cellular aspect; there are usually more cell nuclei in relation to the amount of extracellular fibers, those fibers present are not organized regularly, and the cellular composition is usually more diverse than seen in dense or mucoid CT.

Images to introduce loose CT.

C9: This is a section of the stomach, showing the simple columnar epithelium. Underlying this epithelium you can see the lamina propria, a layer of loose CT containing numerous and differing cells. Note the relative absence of large collagenous fiber bundles.

C10: At a higher magnification, an area of the lamina propria reveals a small blood vessel, different CT cell types including prominent plasma cells, and the collagen fibers.

C15: This whole mount of rat mesentery also shows the collagen fibers of loose CT (here stained yellow) and some different cell types (here stained red). Compare the loose CT on images #C9 and C15 with the dense CT on images #C1, C2, and C3. In image #C15 the animals were fed Trypan blue dye prior to sacrifice. As a result the macrophages phagocytosed the dye and have blue cytoplasmic granules visible.