CONNECTIVE TISSUE OVERVIEW of Connective Tissue Connective tissue

CONNECTIVE TISSUE

OVERVIEW of Connective Tissue Connective tissue forms a framework upon which epithelial tissue rests and within which nerve tissue and muscle tissue are embedded. Blood vessels and nerves travel through connective tissue. • Connective tissue functions not only as a mechanical support for other tissues but also as an avenue for communication and transport among other tissues. Most significantly, connective tissue is the stage for inflammation. The principal cell types involved in immunological defense are found within connective tissue All forms of connective tissue share some common structural features and a common embryonic origin: • Individual connective tissue cells are normally separated from one another by • • varying amounts of extracellular matrix. Cells of connective tissue are not directly attached to one another (unlike epithelial cells). Connective tissue is derived from mesenchyme (unlike most epithelial tissue which is derived from ectoderm and endoderm).

Connective tissue consists of: 1 - Cells: separated by varying amounts of 2 - extracellular substance. The extracellular substance consists of: 3 - fibres which are embedded in ground substance containing tissue fluid. cells in connective tissues, typically account for only a small fraction of the tissue volume. Fibres in connective tissue can be divided into three types: collagen fibres, reticular fibres and elastic fibres.

Cells of connective tissue • • Fibroblasts Histiocytes (tissue macrophages) Mast cells Adipocytes The remaining cell types that you may encounter will be: Endothelial cells of capillaries or Invaders from the bloodstream, particularly lymphocytes and eosinophils.

Fibroblasts are the most common connective tissue cells, have their origen in mesenchyme occur in all types of fibrillar tissue such as areolar tissue. • Resposible for the • • • formation of C. T. fibers and elaborate most if not all the intercellular substance Fibroblasts are elongated and stellate shape The branching processes are long and slender. have abundant basophilic cytoplasm, well developed ER, Golgi complex numerous mitochondria near the nuclei. weakly stained ovoid nucleus with chromatin granules. About an equal number of cells are histiocytes (= macrophages = phagocytes).

Histiocytes (= macrophages = phagocytes • • About an equal number(40% ) of cells are histiocytes (= macrophages = phagocytes). These represent a separate population of cells which are derived from bone marrow. Resting macrophages are difficult to recognize reliably by light microscopy, at least in routine preparations, because they lack conspicuous distinguishing characteristics. They tend to be somewhat larger than fibroblasts, with more cytoplasm. However, after they have consumed something, the cells often can be identified by their cytoplasmic contents without any special stains. One can play a trick with experimental animals to identify macrophages easily in any part of the body. This is to inject a colloid pigment such as trypan blue, carmine or India ink into the peritoneum of a living animal. After a week of injections the animal is sacrificed. Phagocytes all over the body will have picked up the colored material.

mast cell: • These cells are large and • their cytoplasm is distinguished by large numbers of small granules containing heparin and histamine (among other secretory products). With neutral red, these granules are a brilliant red. Mast cells tend to cluster along arterioles (illustration).

Fat cells (adipocytes ) are another important cell type in loose connective tissue. Isolated fat cells or small clusters are mixed in various proportions in many connective tissues. Since most loose connective tissue contains scattered clusters of adipocytes, the term adipose tissue is usually reserved for large masses (grossly visible) of these cells. The most common type of adipocyte is called the unilocular adipocyte or white fat. Each cell contains one single fat droplet surrounded by a thin rim of cytoplasm. • • Conventional techniques of slide preparation use organic solvents so that the fat droplet almost invariably is lost in histological preparations. One sees only a huge empty hole where the fat droplet was outlined with a narrow red band. This appearance has earned them the name of "signet-ring cells". You see nuclei in only a small percentage of fat cells (illustration). This is because the nucleus occupies only a small region and most cells get sectioned in ways that miss that structure. The shape of the droplet, in a tissue section on a slide, depends on how carefully the specimen was prepared. Ideally the droplets are smooth and round (as in the image at right), but they may also be distorted, shaped more like jigsawpuzzle pieces. A curious feature, which you cannot see here, is that each fat cell has its own basal lamina. Yes! Some connective tissue cells have basal lamina in addition to epithelia, whereas many do not (e. g. fibroblasts, macrophages and mast cells). Also, the cells in adipose tissue are supported mainly by reticular fibers, again invisible to the light microscope without special staining.

Brown fat cells “multilocular”: Besides ordinary "unilocular" fat cells seen above there is another variety called brown fat. It gets its name from the color of fresh tissue due to its rich content of mitochondria. Instead of having a single droplet of lipid, brown fat cells are “multilocular”, contain numerous small lipid droplets (hence the name multilocular). The stained cells have a sponge-like appearance with many small holes separated by partitions of cytoplasm. • A collection of tiny fat droplets • • has much more surface area than the single droplet of ordinary fat cells. This allows a much higher rate of metabolism. The main function of brown fat tissue is to produce heat and this tissue is especially prominent in animals that hibernate. Brown fat is scarce in adults but may be found around the adrenal gland. It does occur in babies, around the shoulders and back. (The example here is from an adult monkey. )

Fibres in connective tissue • collagen and reticular fibres: are synthesised by cells of the connective tissues, the fibrocytes. • Elastic fibres are synthesised by both fibrocytes and smooth muscle cells.

Collagen fibres • Collagen fibres are the dominant fibre type in most connective • • • tissues. Arranged in bundles of deferent length and thickness(~10 - 100µm). the fibres are composed of thinner collagen fibrils (0. 2 to 0. 5 µm in diameter). Each of these fibrils is composed of microfibrils, which are only visible using electron microscopy. Axial periodicity with cross bandings at intervals of 64 nm may be visible in thicker fibres by EM. They stain red by Van Geson, pink by H& E. The primary function of collagen fibres is to add strength to the connective tissue.

Reticular fibres • Reticular fibres are very delicate and form fine • • • networks instead of thick bundles. They are usually not visible in histological sections but can be demonstrated by using special stains. For example, in silver stained sections reticular fibres look like fine, black threads (argyrophilic) coarse collagen fibres appear reddish brown in the same type of preparation. PAS ----- magenta

Elastic fibres • Elastic fibres are coloured in fresh tissues - they are light • • • yellow - but this colouration is only visible if large amounts of elastic fibres May occur singly or may be arranged as branches, bundles, or sheets. do not show axial periodecity. Special stains are necessary to show elastic fibres in tissue sections. R esorcin fuchsin is one of these stains, which gives the elastic fibres a dark violet colour are present in the elastic ligaments of the vertebral column and arteries. Also, found in areolar C. T.

Ground substance • is non cellular fibrous material forming the matrix. • found in all cavities and clefts between the fibres and cells of • • connective tissues. It is composed of: 1 - Glycosaminoglycan which mainly contain hyaluronic acid, but also chodroitin sulfates, keratan sulfate, dermatan sulfate and heparin sulfate 2 - tissue fluid: Water, salts and other low molecular substances are contained within the ground substance, but its main structural constituent are proteoglycans. Ground substance is soluble in most of the solvents used to prepare histological sections and therefore not visible in ordinary sections.

Classification of connective tissue Connective tissue proper Supporting Connective tissue Hemopoietic Connective tissue

Connective tissue proper: "Ordinary" connective tissue, or connective tissue "proper", is a generalized form of connective tissue which contains all of the basic components of connective tissue in reasonable proportion, including cells (of several types), extracellular fibers, and extracellular ground substance. Variations in the relative proportions and arrangements of cells, fibers, and ground substance are used to describe/classify connective tissues. In routine use, the term "connective tissue" usually refers to ordinary connective tissue, while the special forms are more commonly referred to by their specific names (e. g. , bone, cartilage, blood). • Composed of two types: 1 - loose connective tissue 2 - dense connective tissue These two tissues are distinguished according to the relative amounts of fibres they contain. • Dense connective tissues are completely dominated by fibres.

Loose connective tissue • is relatively cell rich, soft and compliant. It is also rich in vessels and nerves. • It is best understood as a kind of generalised connective tissue in which all connective tissue cell types may occur.

Types of loose connective tissue: • mesenchyme • Areolar • mucoid • Adipose • reticular

Areolar Loose Connective Tissue: Is the“ideal“ example of ordinary loose connective tissue • refers to the small • • spaces filled with ground substance). Formed from mesenchym has all three types of fibers.

Areolar connective tissue is found: • as part of the dermis of skin (hypodermis), • in the underlying supportive layer (called • • the lamina propria) of the GI, respiratory and genitourinary tracts, also found in serous membranes, and as the body's interstitial tissue. This is the most abundant connective tissue and its matrix contains most of the body's interstitial fluid.

Lamina propria: is areolar connective tissue in the core of the epithelium and beneath the mucusa of the digestive tube • it consist of a delicate network of collagenous and reticular fibers, a few of fibroblasts, mast cells lymphocytes and plasma cells • May highly infiltrated with lymphocytes

tongue, (from rabbit)

The lamina propria in: * the core of the villi * submucosa of the small intestine Loose connective tissue

• The lamina propria: (Yellow arrows)

Areolar connective tissue provides a pathway for all nerves and blood vessels • This slide demonstrates aspects of • • both loose and dense connective tissues after conventional staining with hematoxylin and eosin. The slide is labeled subclavian artery, that being the large circular structure easily visible with the naked eye. It also contains a variety of surrounding structures, all embedded in connective tissue. For example, the clusters of darker pink structures are sections of nerve bundles.

Loose irregular connective tissue (also called areolar tissue). It is rather cellular and supports many small blood vessels which travel through it.

Connective Tissue lamina propria; loose connective tissue

Connective Tissue lamina propria; loose connective tissue

Connective Tissue lamina propria, eosinophils and neutrophils

Connective Tissue lamina propria, eosinophils and neutrophils

Connective Tissue lamina propria, mast cells, metachromatic cytoplasmic granules

Connective Tissue lamina propria, mast cells, metachromatic cytoplasmic granules

Connective Tissue lamina propria, lymphocytes

Connective Tissue lamina propria, lymphocytes

Connective Tissue lamina propria, mast cells

2 - mucous connective tissue, * is a type of mesenchyme found in the embryo * contains much ground substance, stellate and collagenous fibers present in the Umbilical Cord. • Within the umbilical cord you will be able to identify three large vessels and their walls. Mesenchymal connective tissue fills the space between the vessels and the simple squamous epithelium lining the surface of the umbilical cord.

1 - mucous connective tissue, Umbilical Cord, human - H&E

1 - mucous connective tissue, Umbilical Cord, human - H&E

1 - mucous connective tissue, Umbilical Cord, human - H&E

Rticular connective tissue (silvered, black). reticular connective tissue. " It is characterized by very finely divided collagen fibrils, called reticular fibers. Histologists originally defined reticular fibers mainly on the basis of their affinity for "reticulum stains. " This depends upon reactions which reduce silver and is now known to signify unusual quantities of carbohydrates associated with fine Type III collagenous fibrils. • Reticular fibers can look kinky and they commonly give the illusion of branching (although electron microscopy has made it clear that this is not so). • A network of very fine reticular fibers can be seen here, forming the stroma (framework) of a lymph node. These fibers are produced by reticular cells. The pale cells seen in the meshes of the reticular fibers are lymphocytes.

Stellate reticular cells • forming a meshwork of their own cytoplasmic processes. These are in addition to the reticular fiber network which these cells produce -- and which we would see if this tissue were silvered. Notice particularly clear cells in upper left quadrant of field. This slide is from lymph node.

Detail of lymph node, showing stellate reticular cell in middle of field.

Adipose connective tissue • Sometimes considered a specialized connective tissue rather than connective tissue proper, because its cells are closely packed and it has little matrix. • Contains aggregates of fat cells typically clustered in areolar • • • C. T. Also know as fat tissue. Fat does not stain well with H&E and so fat cells will look "empty" -but they are actually full of lipids. Its types: 1 - unilocular (white fat) 2 - multilocular (brown fat) Adipose is found around many organs such as the heart, eyes, kidneys, spleen etc. as well as under the skin and in the medullary canal of long bones. Adipose is also found associated with the serous membranes of the body.

Unilocular adipose tissue (white fat) Contains fat cells filled with a single large droplet of a lipid that contains glycerol esters and fatty acids • Adipose Tissue (Green • • Arrow) A flattened nucleus may be at periphery, giving the cell a “signet ring” appearance. Reticular fibers, fibroblasts, and eosinophils surround these fat cells.

Adipose tissue as seen in a regular histological section. The pale pink tissue mixed in with it is skeletal muscle.

Unilocular adipose tissue often can be found, filling crevices and niches.

Fat tissue is not always well preserved on histological slides. It is easy to allow the delicate rims of cytoplasm of fat cells to partially collapse after the fat has been extracted.

Multilocular adipose tissue (Brown fat) *is a special kind of fat, with small vacuoles in the cells rather than a single large oil drop. * Prominent in the newborn and hibernating animals • Brown fat is also rich in mitochondria. Mitochondria are brown. Can you figure out why? The abundance of mitochondria accounts for the color of liver, kidney, myocardium (mixed with myoglobin for a red-brown), and brown fat.

Dense connective tissues collagenous: (tendons, dermis of the skin, aponeuroses and ligaments). Elastic: yellow legament of vertebral column

They are subdivided according to the spatial arrangement of the fibers in the tissue: • irregular dense connective tissue: the fibers are arranged randomly in all directions • regular dense connective tissue: the fibers are all aligned in a single direction, conferring tensile strength primarily in that direction.

Fibrocollagenous (or just fibrous) tissue • contains a substantial proportion of collagen. A principal feature of • • fibrous tissue is flexibility combined with great tensile strength. Because collagen is colorless and typically scatters light, fibrous connective tissue usually appears white. The sclera (or "white") of the eye is a readily visible example of dense fibrous connective tissue comprising an organ sheath. Tendons and muscle capsule: The ends of muscle fibers are typically attached to dense fibrous connective tissue of periosteum, tendon, or ligament. The dermis of the skin is also fibrous connective tissue (hence, leather is mostly collagen).

Elastic tissue • is a dense connective tissue which contains • predominantly elastic fibers rather than collagen. It is more elastic (obviously) than dense collagenous connective tissue. Examples include the wall of the aorta and the elastic ligament of the spine (called ligamentum flavum [flavum = yellow] because in sufficient quantity elastin is yellowish).

In dense irregular connective tissue: the fibers do not show a clear orientation within the tissue but instead form a densely woven threedimensional network. it forms protective capsules around organs. A good example is the dermis of the skin.

Dense irregular CT (Skin ) The dermis is made up primarily of collagen fibers with very little in the way of cells and matrix. The collagen stains light pink with H&E. In these slides it has a "yellow" appearance. There are fibers running in all directions • Because it is primarily fibers and their orientation is in all planes, the dermis is dense irregular CT. vertical sec.

dense irregular connective tissue This slide shows the dermis located beneath the epithelium. The region where it is mostly fibers and fibroblasts is dense irregular connective tissue. • Much of the dermis of the skin contains bundles of collagen fibers separated by matrix (seen as clear spaces in the slide). • The major cell type found is the fibroblast. These can be seen by their thin black (oblong) nuclei and a thin "stream of cytoplasm" that blends in with the collagen fibers. This is called dense irregular connective tissue because the fiber bundles are organized in an irregular array.

• In regular dense connective tissue the fibres run parallel to each other. Good examples are Tendons, is a strap-like connective tissue that connects muscle to bone. Ligaments and The fasciae and aponeuroses of muscles.

Tendons • Tendons are prime examples. Note that nearly the entire mass of a tendon is composed of aligned, straight bundles of collagen ( illustration). • In van Gieson stained preparations collagen stains dark red while other tissue components appear in varying shades of grey and yellow. • The fibroblasts that originally secreted these fibers are trapped into thin slits between the fibers. These cells are now totally inactive, as indicated by their greatly elongated, heterochromatic nuclei, and are properly called fibrocytes. • Like in most other connective tissues, there will be only a few cells between the fibres. Their cytoplasm will be difficult to identify but the nuclei can be seen scattered among the collagen fibres. Nuclei are often elongated. Their long axis runs parallel to the course of the collagen fibres • Other dense connective tissues, such as ligaments, have less densely packed collagen fibers, but are still regular.

Muscle-Tendon Junction, rat van Gieson

Tendon consists of dense, regular connective tissue. • Collagen fibers are arranged in a parallel manner. The "wavy" pattern is referred to as the crimp. • Note thin, dark staining nuclei of the fibroblasts. Their cytoplasm can not be distinguished.

Tendon consists of dense, regular connective tissue. Note thin, dark staining nuclei of the fibroblasts. Their cytoplasm can not be distinguished.

Connective Tissue dense regular connective tissue, longitudinal

Connective Tissue dense regular connective tissue, longitudinal Dense regular C. T. Parallel bandles of type 1 collagen oriented peripendicular to this arrow Nucleus of fibroblast Dense irregular C. T. Nucleus of fibroblasts

13. Connective Tissue dense regular connective tissue, longitudinal

Connective Tissue dense regular connective tissue, transverse

Connective Tissue dense regular connective tissue, transverse Dense regular connective tissue Skeletal muscle Collagen fibers T. S. Skeletal muscle fibers T. S. Nucleus of skeletal muscle fibers

Dense regular dense connective tissue collagenous • Although the inner part of the arterial wall is made up of smooth muscle, its outer half (its "adventitial" layer) is composed of dense and somewhat regularly arranged connective tissue. One can immediately notice its sudden transition to very loose surrounding connective tissue with a fluffy appearance. In both areas the orange/pink-colored fibers that one sees are collagenous. • It is sometimes difficult to distinguish collagenous fibers from smooth muscle, or even nerve bundles. In such cases, a specific stain must used to differentiate collagen.

Elastin (green arrow) Elastin is a protein commonly associated with connective tissues. This image is of an aorta, a very high pressure blood vessel. The elastin in the aorta enables it to contain blood that is under a large quantity of pressure without exploding.

Objectives You should become able to identify the following in your slide set: • • loose connective tissue dense, irregular connective tissue dense, regular connective tissue reticular connective tissue collagen fibers reticular fibers elastic lamina

• Artery, human - elastin & eosin Like reticular fibres, elastic fibres require special stains to be visualized. Typically elastic fibres will appear as fine, dark violet and gently undulating fibres in the tissue. Elastic fibres can form membranes - not unlike the collagen membrane in the basal lamina of epithelia. This is the case at some levels in the walls of blood vessels. A combination with a second stain is necessary to visualize other tissue components. Colours visible in the sections will depend on the method used in combination with the elastin stain. Eosin gives an even pink or red colour to many tissue components. Nuclei of cells remain unstained without the inclusion of the haematoxylin in the staining solutions.