• General features of connective tissues (CT)
  
• Fibers of CT: collagenous, reticular, elastic
  
• Ground Substance: hyaluronan, proteoglycans
  
• Cells of CT: Resident and immigrant cells
  
• Fibroblasts, macrophages, plasma cells, mast cells, adipose cells, reticular cells, mesenchymal cells
  
• Classification of CT
  
• Embryonic connective tissues: mesenchymal CT, mucous CT
  
• Adult connective tissues: loose CT, dense irregular CT, dense regular CT. elastic CT, adipose CT, reticular CT

The cells found in connective tissues include residents of the respective connective tissues and immigrant cells that have moved in (for example, from the blood). The cells listed here are presented in the following section, with the exception of reticular cells and embryonic mesenchymal cells, which are presented later along with reticular and embryonic mesenchymal connective tissues.

Fibroblasts, which are resident cells, are the most common cells of differentiated connective tissue. They produce the fibers and ground substance of the extracellular matrix. They contact neighboring fibroblasts with their thin processes, and are intimately associated with collagen fibers (their products). Fibroblasts are similar in appearance to mesenchymal cells (shown later). They may be large and somewhat flattened, with branching cytoplasmic processes. It is often difficult to identify the boundary of fibroblasts because the peripheral cytoplasm stains very poorly and the cell membrane is thin and not readily detected. In LM, fibroblasts are most easily viewed in thin, whole mount spreads of connective tissues rather than in sections (in sections, the pale-staining ovoid nucleus is the most prominent feature). Fibroblast shape reflects its environment. For example, fibroblasts are highly compressed in the tendon. Fibroblast morphology also reflects cell activity. In its active state, the cell and its organelles are enlarged. The nucleus is larger, with prominant nucleoli, and the cytoplasm is basophilic and more deeply stained than in the inactive state. Electron microscopy reveals extensive RER, numerous free ribosomes, and a prominent Golgi complex. The relatively inactive form of a fibroblast is referred to as a fibrocyte.

Macrophages are the next most common class of differentiated connective tissue cells, found in all loose connective tissues. Macrophage means "big eater." These cells function in the body's defense mechanisms. Free, wandering macrophages are highly phagocytic, amoeboid cells derived from precursor cells of the bone marrow. In the bone marrow, precursor cells divide and give rise to monocytes. Monocytes circulate in the blood, and may migrate from the blood into the surrounding connective tissue. In the connective tissue, they mature and are then known as macrophages. Macrophages are best identified when actively phagocytic, because ingested particles may then be viewed in the cytoplasm. Macrophages ingest foreign materials, such as dead cells (i.e., neutrophils), tissue debris, including connective tissue fibers, bacteria, surgical sutures, etc. Some of the phagocytosed material is digested by lysosomes, and some is stored indefinitely in the cytoplasm. When the free macrophage becomes highly phagocytic, the entire cell becomes larger, with a larger nucleus. A fixed macrophage is a resting, or inactive cell and difficult to identify. The lifespan of a macrophage varies in different tissues, but may be several months or more.

Macrophages increase in number in areas of inflammation. The increase probably results from the division of cells already in the area, the activation and migration of fixed macrophages from other areas, and the transformation of monocytes that leave the blood and enter the connective tissue. In areas of infection, neutrophils from the blood engulf bacteria. The dying and dead neutrophils are then phagocytosed by macrophages. Pus is composed of dead and dying neutrophils, proteolytic enzymes released by the neutrophils, and tissue debris being digested by the enzymes. Macrophages participate in immunological responses in several ways. For example, they ingest and store antigens, and pass specific information along to other immunocompetent cells. In response to large foreign substances and certain relatively resistant microorganisms, macrophages can fuse to form multinucleated foreign body giant cells.

Macrophages and a number of other specific phagocytotic cells listed here comprise the mononuclear phagocytic system.

Lysosomes are organelles that have phagocytic, degradative functions in all cells and are prominent in active macrophages.

If the tissue is incubated with India ink, colloidal carbon, or trypan blue, macrophages actively ingest these substances, and can be easily identified on the basis of their supravital staining (see virtual slide of Liver and spleen phagocytosis of ink, Odd-018).

Macrophages in red pulp of the spleen are active in phagocytosing dead and dying red blood cells. The are easily identified because of the color shifts that occur in the macrophage cytoplasm during breakdown. This is seen most clearly on the Spleen virtual slide, Odd-047, as indicated here.

Macrophages function in all loose CT, shown here in the active mammary gland.

Plasma cells are not typically in most connective tissues, but are frequently in the connective tissues of serous membranes, digestive and respiratory mucous membranes, and some lymphoid regions. They produce circulating antibodies that then diffuse through the blood plasma, lymph, and other body fluids. In areas of chronic inflammation, the number of plasma cells increases. The plasma cell is an ovoid cell with a small, eccentrically placed nucleus and basophilic cytoplasm. The pattern of its chromatin gives the nucleus a characteristic cartwheel or clock-face appearance. A lightly-stained, perinuclear region of the cytoplasm can be noted that corresponds to the region of the well-defined Golgi complex. Electron microscopy reveals the presence of a very extensive RER, the perinuclear Golgi, and the cartwheel pattern of chromatin in the nucleus.

Plasma cells differentiate from B-lymphocytes. In contrast to the plasma cell, the activated B-lymphocyte (lower panel) has mainly free ribosomes and little RER. The plasma cell shown in the previous slide represents a differentiated end point in its lineage. It does not divide further and survives about 2 weeks, actively producing immunoglobulins during this time.

Plasma cells are easily found on the duodenum virtual slide, Even-013, in regions comparable to that shown on the left. Note that the plasma cells (arrows) have eccentrically placed nuclei, basophilic cytoplasm and a lightly-stained, perinuclear region of the cytoplasm, which corresponds to the Golgi region.

Plasma cells function in other CTs, as shown in the CT of an active mammary gland.

Mast cells are infrequently found in most loose connective tissues, but can be numerous in small groups next to small blood vessels. Mast cells produce heparin, an anticoagulant, and histamine, a substance that causes vasodilation and increased permeability of capillaries. They also contain neutral proteases, eosinophil chemotactic factor of anaphylaxis (ECF-A), and produce leukotrienes (also called slow reacting substance of anaphylaxis, SRS-A). Mast cells participate in anaphylactic shock and certain drug reactions. They are large, oval cells with coarse granules containing these substances densely packed in the cytoplasm. The granules are metachromatic -- they stain dark purple with basic blue dyes and a lavender/pink color with H&E -- because of their high proteoglycan content. In routine sections they are not easily identified because the granules are water-soluble and seldom preserved (slide Odd-006 has good examples). Even when the granules are preserved, the mast cell plasma membrane is frequently ruptured and granules may be seen in the surrounding tissue. Mast cells are easily viewed in connective tissue spread preparations (virtual slide Mesentery with mast cells, Even-006C).

Mast cells divide and are long-lived. They are derived from stem cells of the bone marrow. Mast cells and blood basophils (presented later with blood) are derived from different stem cells, and are not two forms of the same cell. Connective tissue mast cells and mucosal mast cells are distinct; in mucosal mast cells, chondroitin sulfate is found in place of heparin, and the two mast cell types differ in their response to pharmacologic agents.

When a mast cell is activated by binding of antigens and crosslinking of IgE-receptor complexes on the plasma membrane, a signaling cascade is initiated. The result is the release of pharmacologic molecules stored in granules (histamine, ECF, heparin and others) and the activation of phospholipases, which results in the secretion of leukotrienes and prostoglandins.

Stored chemical mediators are released from granules into the surrounding connective tissue very rapidly, as illustrated in the "before" and "after" electron micrographs shown here.

Mast cells (arrows) are easily located in tissue spread preparations; see virtual slides Mesentery with mast cells, Even-006C and Areolar Connective Tissue, SCPM005.

The above guide indicates the connective tissue region just next to skeletal muscle on the slide of developing jaw, Odd-006, where good examples of mast cells (arrows) are located.

Blood leukocytes (white blood cells) are normal components of some connective tissues. Although leukocytes are transported by the blood, they leave blood vessels to perform their functions outside the blood in the surrounding connective tissue. Specific leukocytes, which include monocytes, lymphocytes, and granulocytes (e.g., neutrophils, eosinophils, basophils) are presented in greater detail under blood.

Fat cells, or adipocytes are very large (up to 200 μm in size) and contain major lipid inclusions. In mature adipocytes of white, or unilocular fat, the nucleus and cytoplasm are pushed to the periphery and appear as a thin rim surrounding the lipid droplet(s), resulting in a "signet ring" appearance.

The fat is usually dissolved in routine sections, leaving a large, empty vacuole. The loss of lipid leads to a "chicken wire" appearance. However, the fat can be stained by special procedures using Sudan black, Sudan red (right panel), or osmic acid. Fat cells appear round when they are present singly or in small groups. In large masses, their shapes are deformed due to mutual pressure.

The precursors of adipocytes resemble fibroblasts and are derived from mesenchymal cells. During development and during refeeding after a fast or weight loss, mature fat cells accumulate small lipid inclusions, which then form larger, but still numerous inclusions. These further coalesce until a large, single lipid droplet occupies almost the entire cell. The size of the cell also increases during this process. The fat content is not static; it is continually mobilized and reformed by the processes of lipolysis and lipogenesis. With weight loss, fat cells of white adipose tissue can revert to a precursor form, but retain the potential to again become active fat cells. Thus, the cells become smaller, contain less lipid, but are not lost.

Note the relative size relationship of adipose cells to the neutrophil.

Brown, or multilocular fat cells are smaller in size, and contain several fat droplets rather than one very large one. As a result, the nucleus is not pushed to the periphery. They also contain more mitochondria.

A close relationship is maintained between adipocytes and capillaries in the tissue, which facilitates the ongoing exchange between fat and the blood.

The storage and release of lipid by adipocytes is a dynamic process. Lipids are transported through the blood as chylomicrons and VLDL and hydrolyzed to fatty acids and glycerol by lipoprotein lipase, which is made by the fat cell and transported to the capillary lumen. Fatty acids diffuse through the CT into the fat cell, where they are reesterified into triglycerides for storage. Triglyerides are mobilized by hydrolysis into fatty acids and glycerol via a hormone-sensitive lipase as needed, whereupon the fatty acids and glycerol diffuse through the adipose CT and reenter the blood where they bind albumin and circulate.

A summary of adult CT cells and their functions. Reticular cells and mesenchymal cells are included on the list and will be presented under specific connective tissues (reticular CT and embryonic mesenchyme, respectively).

All adult CTs develop and differentiate from undifferentiated mesenchymal stem cells. Similarly, hematopoietic stem cells give rise to mature blood cells and their derivatives (such as macrophages, mast cells and plasma cells).

All connective tissues have blood supplies, but the abundance of the blood supply varies in the different connective tissue subtypes. Usually, the looser, more cellular connective tissue types have a more abundant blood supply. In the dense connective tissues, the blood supply is less abundant, in some cases, even sparse. This correlation probably reflects the fact that denser connective tissues are primarily composed of non-living fibers and contain relatively few cells.

Connective tissues are generally capable of repairing tissue losses. In some cases, repair processes follow the sequence of embryonic histogenesis. Fibroblasts are principally involved in tissue repair. They function in the repair of connective tissues, and also in the repair of other tissues that have little regenerative capacity. For example, the repair of degenerated cardiac muscle involves replacement by a connective tissue scar.

Reserve mesenchymal cells may proliferate and differentiate into fibroblasts or other cells, functioning in a stem cell capacity. The first fibers to be deposited are fine, and gradually become coarser.

Mesenchymal Connective Tissue: During early embryonic development, the three germ layers of ectoderm, endoderm and mesoderm are defined by the process of gastrulation. The mesoderm, or middle layer, largely consists of a loose arrangement of cells separated by considerable intercellular spaces. This intermediate layer provides support tissues and general "packing material" of the embryo. With time during further development, the cells of the embryonic mesenchyme differentiate into a variety of specialized tissues that include the adult connective tissues and specialized connective tissues.

Characteristic features: Mesenchymal cells are spindle or stellate shaped, and characterized by large euchromatic nuclei and minimal amounts of cytoplasm. The intercellular ground substance is rich in hyaluronan and water. It is homogeneous and relatively fluid, and no extracellular fibers are observed.

Mesenchyme is located in the tooth pulp (see virtual slide of tooth, developing, Odd-0068). Note mesenchymal cells and cell processes and blood vessels (arrow, with RBCs).

Mucous Connective Tissue is a transiently occurring tissue. This form of connective tissue is characteristic of umbilical cord -- Wharton's jelly (see virtual slides of umbilical cord, Odd-010 and Even-009). There, it gradually becomes a more gelatinous, fibrous mass. It is also present in the nucleus pulposus of intervertebral disks and pulp of young teeth.

Characteristic features: Cells of mucous conective tissue are few in number and most similar to mesenchymal cells. The ground substance is very viscous and the tissue "slimy" due in part to a high concentration of hylauronan. Collagenous fibrils and fibers are minimal but increase in quantity and coarseness with time.

Adult Connective Tissues may be classified according to the following subcategories: 1. Loose (areolar); 2. Dense Irregular; 3. Dense Regular; 4. Elastic; 5. Adipose; 6. Reticular. Do not be concerned that different texts and atlases organize the classification schemes somewhat differently. If you consider the breakdown carefully, you will note that the same groups of connective tissues are present in each case. Subtle gradations in connective tissue characteristics make it difficult to strictly classify connective tissue types. This feature of connective tissues is related to the common mesenchymal origin of the adult connective tissues.

Loose (Areolar) Connective Tissue is widely distributed. By definition, "areolae" refer to small spaces. At a gross level, loose connective tissue appears fine, whitish, cobweb-like and stretchy. It is found around muscles, blood vessels, nerves, and organs. It fills spaces and provides an internal framework or stroma for many organs. It contributes to superficial fascia, and underlies many epithelial sheets and basement membranes.

Characteristic features: Areolar connective tissue has a greater variety of cells and fibers than any other connective tissue. Most types of connective tissue cells are found, but the degree of cellularity is highly variable. Fibroblasts and macrophages are most common. Fat cells, mast cells, plasma cells, and several types of leucocytes are also represented. Collagen fibers are most abundant, and usually fine and interwoven. Elastic fibers may be present, but are visible only when they are counterstained. Reticular fibers are found at interfaces of areolar tissue and other organs, or around blood vessels, nerves, etc., but are observed only after special staining. Spaces are filled with fluid-like ground substance. It is useful to observe tissue spreads (left panel) as well as sections of areolar tissue (right panel). Artifactual shrinkage during tissue preparation may reduce the ground substance-filled spaces and cause the loose connective tissue to appear dense.

Functions: Areolar tissue provides support, and, at the same time, its extensibility permits movement of structures. For example, it allows for mobility of the epithelial lining of the intestine where it is present at deeper layers. It loosely anchors nerves, blood vessels, etc. The ground substance of the areolar tissue provides a mechanism for the transport of gases, nutrients, wastes, etc. between capillaries and other tissues such as the avascular epithelia. Defense mechanisms include the function of ground substance as a barrier against the spread of bacteria, and phagocytosis by macrophages and neutrophils. During wound healing, fibroblasts function to repair tissue or form scar tissue. Fibroblasts increase in numbers in areas of active repair. Myofibroblasts may be involved.

Dense Irregular Connective Tissue is characterized by densely packed, abundant fibers (in contrast to loose connective tissue). The fibers are interwoven irregularly to withstand forces exerted from different directions. In contrast, the fibers of dense regular connective tissues are oriented to withstand tension exerted in one direction (later slide). Dense irregular connective tissue includes dermis, capsules of many organs, some deep fascia, dura mater and sometimes the periosteum. On a relatively gross level, these connective tissues appear as interwoven, tough dense masses or feltwork and often appear whitish. Overall, the tissue is characterized by large bundles of interwoven coarse collagenous fibers that are found to be cut in different planes. Some elastic and reticular fibers are present, but are usually recognized only after specific staining for these constituents. The tissue is sparsely populated by cells that include fibroblasts and macrophages, and that may include other connective tissue cells as well. It is difficult to identify specific cells in this connective tissue because the cells are crowded between fibers. Sometimes the tissue falsely appears to have spaces because of shrinkage artifacts during specimen preparation.

Dense Regular Connective Tissue is found in ligaments, tendon, and aponeuroses. Most ligaments are collagenous, or white. On a gross level, this type of connective tissue is seen as large, often whitish, dense cord-like or sheet-like structures. Dense regular connective tissue is characterized by closely packed, parallel groups of very coarse collagen fibers. The collagen fibers are arranged in very regular patterns, formed as a result of forces exerted in the same direction, such as at insertion points where tendon provides the connection between muscle and bone. This connective tissue offers great resistance to tractional forces. The fibers may appear wavy. Fine elastic networks are sometimes present. Usually, fibroblasts are the only cells present, and they appear highly compressed by the tightly packed collagen fibers. Tendon has the most regular arrangement. The collagenous fibers of the tendon are large, and form primary tendon bundles. Together, a variable number of primary tendon bundles are grouped together as fascicles. The tendon fibroblasts are located between the primary tendon bundles and are compressed by them. In longitudinal sections, the cells appear elongated and aligned in rows. In cross section, the cells appear stellate, and they extend thin, radiating processes between adjacent tendon fibers. In the light microscope, sometimes only the nuclei are seen stained.

Elastic Connective Tissue is found in a few ligaments (for example, the ligamenta flava that connects the vertebrae, suspensory ligament of the penis). It is similar in appearance to white (collagenous) ligaments, but is yellowish in color. The elastic fibers are more or less parallel, and branch and anastomose freely. The spaces are occupied by sparse and very fine collagenous fibers and sparsely distributed fibroblasts. The fine collagenous fibers are often coiled. Here, the elastin fibers predominate and are very coarse. For this reason, these elastic fibers can usually be demonstrated even with H & E (top panels). An elastic ligament specifically stained black for elastin is shown in the bottom panels for comparison (SCPM 007).

Adipose Tissue functions include: nutritive (fat actively turns over and serves as a source of energy); protective (soft, shock-absorbing pads); insulative; and cosmetic (softens certain angles of the body). It is widely distributed, and abundant beneath the skin, around the kidneys and heart, in some mesenteries, in the yellow bone marrow, and in the orbit. It is notably absent from eyelids, central nervous system, and lungs. Adipose tissue is atypical among the connective tissues because the cells rather than the extracellular matrix make up the bulk of the tissue. Fat cells predominate in adipose tissue, with a scattering of other connective tissue cells such as fibroblasts and macrophages. These latter cells are often difficult to recognize because they can be highly compressed by the presence of the neighboring fat cells. The fat cells are surrounded by delicate strands of reticular, collagenous, and elastic fibers. Adipose tissue is richly innervated and highly vascularized; each fat cell is in contact with at least one capillary. Adipose tissue is of two types.

White, or unilocular adipose tissue is most common. White fat cells contain one central fat droplet in their cytoplasm, leading to the characteristic "signet ring appearance". Often, unilocular adipose tissue is organized into small lobules by connective tissue septa. The tissue has a "chicken wire" appearance in routine sections where the fat is lost. Obesity in adults may result from excessive accumulation of fat in cells that become larger than usual or from an increase in the number of adipocytes. An increase in the numbers of adipocytes occurs during the early weeks of life, and may be caused by overfeeding.

Brown, or multilocular adipose tissue is composed of cells containing numerous lipid droplets and mitochondria. It is located in several specific places in the human embryo and newborn, remains restricted to these regions after birth, and is greatly reduced in the adult (after birth, no more brown fat is formed). It functions mainly in the first few months of prenatal life to produce heat and protect the newborn against the cold. Brown fat is commonly found in hibernating animals.

Reticular Connective Tissue provides a fine support--the definition of reticular is "net-like". Reticular tissue forms the fine stroma of lymphoid (lymph node and spleen) and myeloid (bone marrow) organs, providing a loose support for populations of cells.

Reticular connective tissue is characterized by an an open-structured reticulum composed of reticular fibers and reticular cells (arrows, inset). It is the connective tissue support in specific, restricted regions such as hematopoietic organs (lymph node and spleen). Other cells of these organs, including lymphocytes and macrophages, are free to pass through the open spaces of the reticular tissue. When the spaces are crowded with lymphocytes and other cells, the reticular cells and fibers seem largely inconspicuous. Reticular cells are most similar in appearance to mesenchymal cells. Their most prominent features are large, ovoid, pale-staining nuclei. Reticular fibers are not evident with routine staining (compare right (H&E) and left (silver stained) panels). After silver staining, the fine, argyrophilic fiber network is easily observed, but usually the cells stain poorly, or not at all. The relationship of reticular fiber support to the populations of cells is beautifully represented in the Lymph node, reticular stain, virtual slide Even-0448 shown in the bottom panel, where the cells are also very well-preserved and stained.