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Lab 2. Connective Tissue

Connective tissue (C.T.) is the second of the four primary types of tissue in the body. It includes:

  • Loose or areolar connective tissue
  • Dense connective tissue
  • Specialized connective tissue
  • Adipose tissue
  • Blood cells
  • Bone
  • Cartilage

In this laboratory session we will study loose and dense C.T., adipose tissue and cartilage. Students should aim to distinguish the different types of connective tissue and understand the role of their cellular, fibrous and ground substance components.

Connective tissues consist of cells and intercellular material. Fibroblasts, adipose cells, mast cells, macrophages, leukocytes and plasma cells are present in loose connective tissue. The intercellular substance (matrix) consists of fibers (collagenous, elastic, reticular), amorphous ground substance (glycoproteins, proteins, proteoglycans) and tissue fluid. Each type of C.T. has its own characteristic pattern and proportion of intercellular materials and cells; you should observe this carefully while studying your slides. The object of this laboratory session is to introduce you to loose and dense connective tissue, so that you can distinguish them, identify their various morphological components, and understand their structure. Loose or dense C.T. is present in virtually every slide in your box, and you should learn to observe the types of C.T. in different organs and think about the functional roles they play in these different settings.

Loose (or Areolar) Connective Tissue

Slide #7 is not a section of tissue; it is a piece of mesentery cut from a larger sheet, and each piece was processed individually. Consequently, more variation exists from slide to slide than with sections from a single block. Mesentery is organized as a "sandwich" of connective tissue between two simple squamous epithelial layers (see figure). The slide was stained with H & E and also resorcin fuchsin (to stain elastic fibers) and neutral red to stain mast cell granules.

Examine slide #7 with the 4x objective. The tissue will appear unevenly stained, indicating uneven distribution of tissue components. In the best preparations, dark maroon dots will be seen in the tissue. These are the nuclei of various cell types present in the mesentery. With the 10x objective some of the deep maroon dots observed will be larger and may be recognizable as granular cells called mast cells. Large, round light spaces if present are fat cells. The fibrous character of the tissue should be obvious. Clearly visible are elastic fibers which are very fine and stain deep purple. The collagen fibers are wider and stain pink. Reticular fibers are not discernible with this stain. At higher magnification (40x objective), unevenness of the mesentery will be apparent. Several areas may be in focus and several areas out of focus in the same field. Collagen fibers are seen as straight or wavy pink bands of varied thickness. They are made of smaller units called fibrils, which may be seen with the condenser diaphragm almost closed. In this respect, collagen fibers resemble a cable made up of many individual parallel strands. The fiber bundles sometimes branch and anastomose, forming a network, but individual strands (fibrils) do not branch. See your textbook for the E.M. appearance of collagen with the characteristic periodic cross-striated structure.

The elastic fibers of loose C.T. have a much smaller diameter than the collagen fibers, but are larger than the individual collagen fibrils. Under the light microscope, the elastic fibers are morphologically homogenous. They are not usually wavy. They are composed of microfibrils and elastin, a complex protein which does not stain well with ordinary stains. Unless specially stained (e.g., orcein or resorcin fuchsin) elastic fibers are difficult to distinguish. In electron micrographs (see your textbook) elastic fibers are seen to consist of two-components — an amorphous inner component called elastin and peripherally arranged microfibrils, about 10 nm in diameter.

The largest nuclei visible on this slide are those of the simple squamous epithelial or mesothelial cells and the cytoplasm of these cells is not visible. The next largest oval nuclei are those of fibroblasts. Some of these cells may have a pale pink cytoplasm, but most will not exhibit cytoplasmic staining. The large cells with closely packed granules are mast cells. In preparations which were left too long in hypotonic solutions during processing the mast cells became swollen and burst and the granules may be scattered around. A few polymorphonuclear leukocytes and lymphocytes from the blood may also be present. These have lobulated or dark, round nuclei, respectively, and will be studied in detail later in the course.

The ground substance of the connective tissue, which would occupy the space between the fibers and cells, is pale staining, and much of it is lost during most histological preparations. Special methods of preparation are needed to preserve this important component of connective tissue. Return to low power for an overall impression of the structure of the tissue. On some of the slides, blood vessels may be seen. The larger ones are more deeply stained than the connective tissue through which they pass. The small blood vessels (capillaries) are the size of collagen fibers, but may be distinguished from the latter by the presence of thin nuclei (of endothelial cells) oriented parallel to the axis of the capillary. Capillaries may contain closely packed red blood cells, or they may be collapsed and contain no blood.

Connective Tissue Macrophages

Macrophages play an important role in connective tissue, by ingesting or phagocytizing foreign matter and bacteria. However, they are difficult to discern in normal preparations, and their abundance in the connective tissue is not obvious. Special techniques can be used to label macrophages, especially injection of dyes, particles of which are then ingested. This allows for visualization of macrophages and their distribution.

Adipose Tissue

Where C.T. is composed predominantly of adipose (fat) cells, it is called adipose tissue. Adipose tissue is found in the panniculus adiposus, in bone marrow, and around the kidneys, but none is found in the central nervous system. Examine the pale-staining adipose tissue located between muscle and bone in slide #25. When packed together, these cells give the appearance in ordinary histological preparations of chicken wire (i.e. an open network of fine, pale pink-staining material enclosing roughly circular open spaces). In living tissue, these vacuoles in the cells are filled with fat and the shape of the cell is nearly spherical. In the preparation of histological sections, the fat is dissolved out. These cells may be as large as 120 micrometers in diameter. Single adipose cells may be seen in the bone marrow (the part of the section that looks purple at low power). Under high power, the fat appears to compress the surrounding cytoplasm into a thin layer. The nucleus is flattened and pressed against the cell membrane giving a signet ring appearance. Between the adipose cells may be seen some flattened fibroblast nuclei and capillaries.

Dense Connective Tissue

These tissues are characterized by an overwhelming preponderance of the fibrous component of the tissue, with a correspondingly smaller volume of ground substance, tissue fluid and cells. Collagenous fibers are characteristic of dense, white C.T. whereas elastic fibers are characteristic of dense yellow C.T. The fibers may be either regularly or irregularly arranged.

Irregularly Arranged Dense Connective Tissue

Examples of this tissue are the deep fascia of the body, the capsules of some organs, the dermis, periosteum and perichondrium. This tissue usually occurs in the form of sheets or membranes in which the fibers interlace and form a dense tough feltwork. The arrangement is not completely irregular, because many of the fibers are approximately parallel to the surface.

Examine the dermis of the skin, slide #43. The layer underlying the epithelium is dense irregular C.T. This pink-staining tissue is composed mainly of thick interlacing collagenous fibers. Spaces between fiber bundles are artifacts which arise during sample preparation. Nuclei of fibroblasts can be seen between the bundles, but the cytoplasm of these cells is not easily discernible. Compare and contrast these cells with the fibroblasts of loose C.T. Cells other than fibroblasts may be present. (Sections of sweat gland ducts and blood vessels which may also be seen will be studied at a later date).

Regularly Arranged Dense Connective Tissue

Examples of this type of C.T. are tendon, ligament and aponeurosis. The collagen fibers are arranged parallel to each other, giving the tissue great tensile strength along the direction of stress. Examine a longitudinal section of tendon, slide #10 (odd boxes). The tendon stains pink and the wavy appearance is due to lack of tension at the time of fixation, and the uneven staining may be related to the way in which the wavy fibers were cut. In some slides, the attachment of the tendon to striated muscle is seen. The tendon is continuous with the dense connective tissue which ensheathes the muscle fibers (epimysium). The nuclei of fibroblasts within the tendon are very long and slender, lightly stained, and may often be seen to lie in rows between the bundles of collagen fibers. The cytoplasm is usually indistinct. The shape of these fibroblasts is modified by the orientation and pressure of surrounding collagen fibers.

Dense Elastic Tissue

This tissue has a preponderance of elastic fibers. Examine the longitudinal and cross sections of ligamentum nuchae (ungulate) on slide #10 (even boxes). This is an elastic ligamentprepared with resorcin fuchsin which stains elastin purple. It is very difficult to distinguish the cells. Observe that the tissue consists of parallel bundles of thick elastic fibers held together by loose C.T. containing some blood vessels. Collagen fibers stain pink. The arrangement of elastic fiber bundles and loose collagenous connective tissue is more easily seen in the cross section. Note that these elastic fibers are much thicker than those of loose C.T. Most other ligaments do not have a preponderance of elastic fibers, and are similar to tendon in composition, although not as regularly arranged.

Reticular Connective Tissue

Reticular fibers consist of one or more types of very thin collagen fibers, mostly types with which abundant carbohydrate has been combined. Thus, they react with silver stains and with periodic acid-Schiff reagent but are not demonstrated with ordinary histological stains. Reticular C.T., containing a 3-dimensional network of reticular fibers, make up the stroma of lymphatic and hemopoietic tissues such as lymph nodes, thymus, spleen and bone marrow.

Examine a section of lymph node stained by a silver impregnation technique, slide #34, and observe the black staining framework of reticular fibers. The fibers makeup a honeycomb-like matrix in which cells (mostly lymphocytes) take up residence. The round structures formed by some of the reticular fibers are lymphoid follicles, centers of imune activation. The cells may be seen on some parts of the slide where the nuclei may have taken up some of the stain, but most of the cells are unstained. What is the pink-staining material which is interspersed within the reticular framework? Note the variations in the architecture of reticular fibers which you will come to understand later on.

 

Cartilage

OBJECTIVES

Students should aim to recognize and differentiate the 3 types of cartilage, understand the composition of the extracellular matrix and the role of chondrocytes in producing and maintaining it.

Cartilage is a specialized form of connective tissue containing chondrocytes which secrete, and are surrounded by, an extensive extracellular matrix (ECM).  Chondrocytes occur singly or in isogenous groups, composed of 2-8 cells derived by mitosis from a single chondrocyte and locked in by the relatively stiff matrix.  The cells are seen to be in the lacunae (cavities) within the ECM.  These apparent spaces are artefacts of shrinkage but are characteristic of chondrocytes and thus cartilage. The strength and durability of cartilage are properties of the matrix, which is an interlaced network of collagenous and/or elastic fibers in a ground substance, a hydrated gel of complex proteoglycans.  The collagen is mostly Type II.

There are three types of cartilage characterized by the composition of the extra-cellular matrix.  (1) hyaline, (2) elastic and (3) fibrocartilage.

Hyaline cartilage is found lining articular surfaces, in the nasal septum, tracheal rings, costal cartilage and the epiphyseal cartilage of growing bone. Study the trachea on slide #72 (trachea). Even numbered boxes usually contain slides stained for elastic tissues, odd numbered boxes have slides stained with hematoxylin and eosin. Even slides will have a brown coloration, odd slides the more familar pink and blue staining. In this slide considerable fading has occurred, and the normal basophilia of freshly stained cartilage is almost absent. The trachea is a large open tube, with cartilage constituting the principal component of its wall. Using the scanning lens, locate the cartilage rings. At higher power, identify single chondrocytes and locate isogenous groups. There are clear areas between many of the chondrocytes and the walls of their lacunae because of cell shrinkage during histological preparation. Identify the perichondrium. Note the presence of flattened fibroblasts and compare these to the round cells within the cartilage. The perichondrium is acidophilic due to the presence of numerous collagen fibers. The cartilage matrix contains collagen fibers, but these are very difficult to see.

Elastic cartilage is found in the ear and epiglottis, where it provides a rigid, but elastic, framework. Its principal components are elastic fibers and type II collagen. Examine a section through infant ear, slide #11 (even), which has been stained for elastin with resorcin fuchsin. The purple staining interlaced fibers run in all directions, and appear as dots when cut transversely. The nuclei and cytoplasm of the cells are not stained well. The cartilage in this section is immature and therefore richer in cells than adult cartilage. Compare this arrangement of cells and fibers with that of hyaline cartilage in the trachea.

Fibrocartilage is found in intervertebral discs, in menisci of joints, and often where tendon and ligament are joined to bones. It resembles a dense CT which contains islands of chondrocytes embedded in cartilage matrix. Its appearance varies with its location. The major and characteristic intercellular component of this tissue consists of thick, interlaced collagenous fibers. Examine a section of intervertebral disc (slide #12). It is difficult to distinguish from dense CT. However, careful examination will reveal some cells within lacunae. You may not be able to discern the difference between chondrocytes and fibroblasts by nuclear shape, but if a lacuna surrounds the cell, it is a chondrocyte; if not, a fibroblast. There is no perichondrium.