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CHAPTER 3. Connective Tissue

The microscopic study of animal tissues and cells

Overview – Connective Tissue
Plate 3-1. Dense Irregular Connective Tissue 
Plate 3-2. Dense Regular Connective Tissue: The Tendon
Plate 3-3. Reticular Fibers: The Framework Of The Spleen
Plate 3-4. Fibroblasts, Mast Cells, And Macrophages

As human beings, we are often quick to forget that we are organisms – groups of organs held together in such a way that we can walk, talk, live, love, and study microanatomy. We can perform all of these highly specialized functions because our various organs are held together in precisely the right spatial arrangements. The key words here are “held together”; we are held together by groups of extremely efficient and carefully designed cells and tissues collectively called connective tissue.

There are several kinds of connective tissue. All are variations on a common theme, in that they are combinations of cells and extracellular fibers and fluids that are strong, resilient, and capable of repairing themselves. In this chapter, we will examine the various kinds of cells, fibers, and fluids that make up the connective tissues of the human body.

The most common type of cell found in connective tissue is the Fibroblast. A cell that can adopt many shapes, depending on its activity, the Fibroblast produces the two major classes of extracellular material found in connective tissue: fibers and Ground Substance. Fibers are strands of proteinaceous macromolecules that make connective tissues strong, resilient, and elastic; ground substance is a complex, viscous fluid that makes up the Matrix in which the fibers and cells are embedded

The three main types of fibers in connective tissue are Collagenous Fibers, reticular fibers, and elastic fibers. Of these, Collagenous Fibers are the strongest and also the most common. While the strength and durability of leather is well established, few realize that leather consists of interwoven Collagenous Fibers – fibers that originate from the Dermis of the skin of large mammals. Strong though leather is, the Collagenous Fibers from which it is made are even stronger in the living animal. Collagenous Fibers possess a formidable tensile strength; they can withstand pulling forces of up to 300 kg/cm ? ? ? without rupturing. Under those conditions, the Collagenous Fibers will stretch very little – 3% at most. The properties of Collagenous Fibers are derived from the fibrous protein Collagenfrom which they are assembled.

Because Collagenous Fibers stretch so little and connective tissue so often needs to be elastic, the elastic Fiber has evolved. When stretched, an Elastic Fiber can return to its original length. Elastic fibers, which are also made by fibroblasts, have two components-a fibrous core and an amorphous covering. The amorphous covering, made of the protein Elastin, is thought to be responsible for the capacity of elastic fibers to recoil after being stretched. It is no accident, then, that connective tissues subjected to deformation often contain both kinds of fibers, Collagenous Fibers for strength and elastic fibers for elasticity. Perhaps the best-known example of this kind of connective tissue is found in the wall of the aorta, which with every beat of the heart must stretch to withstand tremendous hydrostatic forces and then instantly recover its original shape when those forces are reduced.

The third class of connective tissue Fiber, the reticular Fiber, is apt to confuse beginning students of microanatomy. Before the advent of electron microscopy, histologists had identified a class of connective tissue that consisted of fine fibers, thinner than Collagenous Fibers, that had special staining qualities. These fibers, often distributed in a spiderweb or “reticular” fashion, were called reticular fibers after their configuration. They are common to organs of the immune system, in which they form the connective tissue framework for the spleen and lymph nodes. More recently, electron microscopists have shown that reticular fibers are actually made of Collagen fibrils. (A Fibril is a tiny structure, visible by electron microscopy but too small to be seen by light microscopy. Many fibrils, when packed together, make up a Fiber). Within a given reticular Fiber, the Collagenfibrils that compose its core are covered by a Glycoprotein coat that resembles material of the Basement Membrane. In organs such as the spleen and lymph node, the reticular fibers are surrounded by thin cytoplasmic extensions of reticular cells – cells, quite similar (perhaps identical) to fibroblasts, that are thought to elaborate the reticular fibers. In ordinary histologic preparations not specially stained to detect them, reticular fibers are impossible to distinguish from Collagenous Fibers.

Connective tissue has been classified into several categories based on the manner of packing of the fibers and the ratio of cells to fibers. Connective tissue that features many densely packed fibers going in many different directions is known as dense irregular connective tissue. Found in such places as the Dermis of the skin and the Submucosa of the gut, dense connective tissue is well suited to binding epithelial sheets to underlying tissues.

Connective tissues that are subjected to the exertion of heavy forces in one direction, such as the forces that pull on tendons and ligaments, are quite different. They have densely packed Collagenous Fibers oriented parallel to one another. This type of connective tissue, known as Dense Regular Connective Tissue, has many fibers and relatively few cells. The paucity of cells, most of which are fibroblasts, may account for the slow healing of torn tendons.

Connective tissue in which there are relatively few fibers and lots of cells is loose connective tissue. Found in such places as the Lamina Propria of the gut, loose connective tissue is composed of fibers that run in many directions and form an open, watery, cellular meshwork. Loose connective tissue often contains collagenous fibers, elastic fibers, and reticular fibers woven together in a variety of ways.

Connective tissue cells are varied. Most abundant is the Fibroblast, which seems to be capable of secreting all of the types of fibers. Mesenchyme cells, too, are abundant. Mesenchyme is embryonic connective tissue; Mesenchyme cells, therefore, are embryonic connective tissue cells. Despite their embryonic nature, Mesenchyme cells persist in the connective tissues of the adult. Similar in appearance to undifferentiated fibroblasts, these Mesenchyme cells are capable of developing into whatever type of connective tissue cell is required at the moment. They are thought to be multipotent and can differentiate into fibroblasts, cartilage cells, bone cells, and, on occasion, smooth muscle cells.

In addition to Mesenchyme cells, macrophages and mast cells are widely distributed throughout connective tissue. Macrophages are large cells filled with Enzyme-packed lysosomes that can phagocytose (i.e., engulf and digest) unwanted foreign particles and cellular debris. Mast cells, however, are different. Filled with conspicuous large granules containing Heparin and Histamine, mast cells can “degranulate” – often in response to injury – and release Heparin, which slows blood clotting, and Histamine, which increases capillary permeability. In this manner, mast cells participate in the inflammatory response; they promote the flow of blood out of the bloodstream and into tissue spaces, where blood cells and antibodies can fight infection. Chapter 4 will describe the various types of cells found in circulating blood – the so – called red blood cells, or erythrocytes, and the white blood cells, or leukocytes. The white blood cells include, among others, lymphocytes, monocytes,neutrophils, and eosinophils. These so-called white blood cells are actually connective tissue cells; they perform their function in the connective tissues of the body, not in the blood. The bloodstream simply serves to transport them from their point of origin (usually in the bone marrow, lymph nodes, or spleen) to their ultimate destination in the connective tissues. Consequently, microscopic examination of connective tissue often reveals the presence of lymphocytes, monocytes (which are immature macrophages), neutrophils (which are phagocytes), and eosinophils (which phagocytose AntigenAntibody complexes). In addition, cells called Plasma cells are frequent inhabitants of loose connective tissue. Plasma cells, derived from a class of Lymphocyte called the B-lymphocytes, are large cells filled with many Cisternaeof the rough Endoplasmic Reticulum that manufacture and release antibodies that bind to antigens in the course of the immune response.

Plate 3-1
Dense connective tissue is extremely tough. It can be pulled in many directions with considerable force and still maintain its structural integrity. The major reason for its great strength is its high content of collagenous fibers. As described in the overview, Collagenous Fibers are very strong. In Dense Irregular Connective Tissue, they run in many directions and are woven into a compact feltwork that contains relatively few cell

Figures A and B at right are a matched pair of light and electron micrographs of serial sections taken through the Dense Irregular Connective Tissue of the Lamina Propria of the tongue of a macaque. (The Lamina Propria, which consists of Dense Irregular Connective Tissue in the tongue, consists of loose connective tissue throughout the remainder of the alimentary canal.) Light microscopy (Figure A) cannot show where a cell ends and the fibers begin. Under the electron microscope, however, cellular boundaries become clear even at relatively low magnification (Figure B). Consequently, carefully comparing the images in Figures A and B point by point can show which structures underlie the somewhat fuzzy images seen in the light microscope. Take, for example, the Fibroblastencircled in both figures. In Figure A, it looks like a blob that is somewhat darker than the surrounding material. Figure B shows that the blob is indeed a cell, replete with a Nucleus and fine cytoplasmic extensions. Surrounding the cell on all sides are wavy Collagenous Fibers (C) that are collections of tiny Collagen fibrils illustrated later in this chapter.

It is evident from these images that dense irregular connective tissue contains a large number of fibers and relatively few cells. Of the cells, most are fibroblasts – cells that synthesize and secrete Collagen as a large number of tiny protein subunits that are assembled outside the cell into long, thin, Collagen fibrils. In addition to the fibroblasts, a number of other cells common to connective tissue are present in the field of view. In the center of the field is a cross-sectional image of a nerve bundle (N). Within this nerve bundle, some nerves are surrounded by an electron-dense myelin sheath. The surface of the tongue is highly sensitive, and numerous peripheral nerves course through the dense connective tissue beneath the epithelium. In addition, many tiny capillaries (Ca) supply blood to the connective tissue as well as to the overlying avascular epithelium. Mast cells (M), too, are present. These cells are filled with conspicuous electron-dense granules that contain Heparin and Histamine. Mast cells are frequently located in the vicinity of small blood vessels such as capillaries and post capillary venules.

When sectioned material is observed with the light microscope, Dense Irregular Connective Tissue is often seen to contain small, thin, dark streaks that contrast with the surrounding pale-staining Collagenous Fibers. These streaks (arrows) are usually thin cytoplasmic extensions of fibroblasts, whose cell bodies are not in the plane of section. Here again, the identity of these streaks (arrow) will become clear when carefully compared in Figures A and B at right.

Plate 3-1

Figures A and B. Matched pair of light and electron micrographs of serial thick and thin sections of the Lamina Propria of the tongue of the macaque. C, Collagenous Fibers; Ca, capillary; F, Fibroblast; M, Mast Cell; MF, muscle Fiber; N, nerve bundle; arrow, slender cytoplasmic process extending from Fibroblast; circle, the same Fibroblast in Figures A and B. 1,600 X

Plate 3-2

Dense Regular Connective Tissue: The Tendon

The function of the Tendon is to provide a link between muscle and bone and to cause the bone to move when the muscle contracts. The simplicity of Tendonfunction is reflected in its straightforward microanatomy. Tendons and related ligaments, which hold bones together, are made of Dense Regular Connective Tissue that consists of a small population of fibroblasts in a field of parallel Collagenous Fibers.

Figures A, B, and C at right illustrate the fundamental microanatomy of Dense Regular Connective Tissue. Figure A is light micrograph of a longitudinal section through a monkey Tendon. The image appears as a series of dark, parallel streaks in a pale, amorphous background. The dark, parallel streaks are actually fibroblasts (F), or parts of fibroblasts, cut in longitudinal section. The pale, amorphous background consists of hundreds of tightly packed collagenous fibrils (C), all oriented parallel to one another and to the long axis of the Tendon itself.

The microanatomy of the Dense Regular Connective Tissue of the Tendon is more readily apparent when viewed by electron microscopy. Figure B is an electron micrograph of a longitudinal section through the same Tendon shown in Figure A. Here, a lone Fibroblast (F) is visible, sandwiched between the parallel, densely packed Collagenous Fibers (C) it has laid down around itself. Fibroblasts in tendons tend to be flat, and this one has been sectioned at right angles to its broad axis.

Consequently, it appears quite long and extremely thin and seems to have very little Cytoplasm. The most prominent feature of this Fibroblast is the long, dark Nucleus (N). Its large Complement of condensed Chromatin suggests that the cell is relatively quiescent; that is, it is not producing large amounts of Collagen. The Cytoplasm is spread out in a thin envelope around the Nucleus. Immediately around the perimeter of the cell lie Collagen fibrils that, on close inspection, display the periodic cross-banding pattern so characteristic of Collagen.

The fibrillar nature of the Collagen is immediately evident in Figure C, an electron micrograph of a cross section through a Tendon. Here again, as in Figure B, we see a single Fibroblast (F) surrounded by a field of Collagen fibrils (C). In this figure the fibrils, having been cut in cross section and viewed on end, have a distinct punctate appearance. As a result, the image shows a lone Fibroblast‘s Nucleusamidst thousands of more or less evenly spaced dots. The dots, each of which represents a single CollagenFibril, are of relatively uniform size, measuring about 1300 Angstroms in cross-sectional diameter. In the region just beneath the Nucleus, a stray, wavy CollagenFibril has been cut in longitudinal section along part of its length (arrow); its cross-striations are visible even at this relatively low magnification.

connective tissue

Plate 3-2

Figure A. Light micrograph of a longitudinal section through a Tendon from the finger of the squirrel monkey. C, Collagenous Fibers; F, Fibroblast. 500 X Figure B. Electron micrograph of a longitudinal section through the same Tendon shown in Figure A. C, Collagen fibrils; F, Fibroblast; N, Nucleus of Fibroblast. 11,400 X Figure C. Electron micrograph of a cross section through the same Tendon photographed in Figures A and B. C, Collagen fibrils; F, Fibroblast; N, Nucleus of Fibroblast; arrow, part of Collagen Fibril cut in longitudinal section. 13,000 X

Plate 3-3
As described in the overview, connective tissue may contain any (or all) of three kinds of fibers; collagenous fibers, reticular fibers, and elastic fibers. Of these, reticular fibers are perhaps the most difficult to envision. Reticular fibers often coexist with Collagenous Fibers in the same connective tissue and are impossible to identify with the light microscope unless special stains, such as silver stains, are used.

Although widely distributed throughout the connective tissues of the body, reticular fibers perform a crucial function in establishing the connective tissue framework of tissues and organs associated with the immune system. The spleen and lymph nodes, for example, are two organs of a somewhat “mushy” consistency that would melt into a puddle were they not held together by a spiderweb-like framework of reticular fibers. Figures A and B at right are electron micrographs that depict a reticular Fiber caught in cross section as it passes through (and supports) the so-called red Pulp of the spleen.

The reticular Fiber (arrow) consists of three components. First, the core of the Fiber contains a number of individual Collagen fibrils (C) that are presumed to be similar to those found in Collagenous Fibers. Here, they appear as a series of electron-dense dots, much as they did in the cross sectioned Tendon described earlier in this chapter. Second, the Collagen fibrils within the reticular Fiber are closely associated with some dense, amorphous material (A) that resembles the amorphous component of the Basement Membrane. Third, both the Collagenfibrils and the associated dense, amorphous material are surrounded by a thin cytoplasmic extension (R) of a reticular cell. (The reticular cell, often called a Reticulocyte, elaborates reticular fibers).

One of the most dramatic features differentiating reticular fibers from Collagenfibers is that Collagen fibers are “naked” fibers, unlike reticular fibers, which are surrounded by thin envelopes of Cytoplasm from reticular cells. This difference raises a question about the functional significance of the cellular covering of reticular fibers. If the primary role of reticular fibers is structural support, why are they not, like collagenous fibers, naked fibers? The answer to that question is unknown at present. It is interesting to speculate that, because reticular fibers are found throughout tissues and organs of the immune system, perhaps the presence of a reticular cell wrapping on the outside of the reticular Fiber has some functional significance related to the workings of the immune system. It is well known, for example, that cell membranes have many specific receptors on their surfaces. This fact is particularly true of cells of the immune system, which rely on highly specific surface-to-surface interactions with cells and antigens in the normal course of their lives. In organs of the immune system, the reticular fibers provide a framework to which cells of the immune system are attached. Covering that framework with a Cell Membrane – the Cell Membrane of the reticular cell, in this case-provides a receptor rich surface that facilitates the function of the immune cells that attach to that framework. In Figures A and B, a Lymphocyte (L), located at the upper left corner of each micrograph, sits so that its outer limiting membrane is closely apposed against the Cell Membrane of the reticular cell (*), thus establishing a large surface area of contact between the two cells.

Plate 3-3

Figures A and B. Electron micrographs of a reticular Fiber from the red Pulp of the spleen of the macaque cut in cross section. A, amorphous material associated with Collagen fibrils; C, Collagen fibrils; E, Erythrocyte in red Pulp of spleen; L, Lymphocyte; R, cytoplasmic extension of reticular cell; arrow, reticular Fiber; (*) close contact between cell membranes of lymphocvte (L) and reticular cell (R). Figure A, 18,000 X; Figure B, 44,000 X

Plate 3-4
Connective tissue consists of a mixed population of cells embedded in an extensive Extracellular Matrix of fibers and Ground Substance. The cells found in connective tissue are of many types, including fibroblasts, mast cells, macrophages, Plasma cells, and all the cells usually known as white blood cells – i.e., lymphocytes, monocytes, neutrophils, and eosinophils. The white blood cells, which are actually connective tissue cells, will be described in Chapter 4. Here, we will concentrate on three other connective tissue cell types of considerable importance: fibroblasts, mast cells, and macrophages.

In Figure A, the electron microscope reveals several fibroblasts. Fibroblasts typically have large, centrally located, football-shaped nuclei (N). The Nucleus has a peripheral ring of densely stained Heterochromatin (*) and a prominent Nucleolus (Nu). Out in the Cytoplasm and near the Nucleus lies a well-developed Golgi complex (G) closely affiliated with abundant Cisternae of the rough Endoplasmic Reticulum (RER). Taken together, these organelles produce and release molecular precursors of Collagen fibrils as well as the amorphous Ground Substance in which the Collagen fibrils (C) come to be embedded.

In addition to fibroblasts, mast cells are often evident in sections of connective tissue. Usually located near blood vessels, mast cells – one of which is illustrated by electron microscopy in Figure B – are large cells of irregular shape. The most prominent feature of the mast cell, aside from the Nucleus (N), is its large content of electron-dense granules (G) that occupy the bulk of the Cytoplasm. These granules contain Heparin, an anticoagulant, and Histamine, which increases capillary permeability. When released from the Mast Cell, these substances promote the flow of blood from small vessels into connective tissue spaces at sites of infection or injury, thereby facilitating the process of inflammation essential for proper healing.

Connective tissue also contains a large number of highly specialized macrophages, which are large cells capable of Phagocytosis. During the process of Phagocytosis, a Macrophage will detect unwanted material such as bacteria and cellular debris, send out a pseudopodial extension of its Cytoplasm, and engulf the foreign matter. The engulfed matter, drawn into the Macrophage‘s own cell body, is subsequently digested by enzymes contained within the Macrophage‘s many lysosomes. Figures C and D at right are electron micrographs that depict macrophages in action. In Figure C, a Macrophage (M) is shown engulfing and digesting an aged red blood cell (E) that has escaped from the leaky, blood-bearing sinusoids of the spleen. Here, the Nucleus (N) of the Macrophage is visible, as is the bulk of its cell body. The lysosomes (L) that carry the lethal digestive enzymes are abundant. This Macrophage has squeezed itself between the reticular fibers (R) that form the connective tissue framework of the spleen. When the Macrophage has finished digesting the Erythrocyte, it will recycle its components for further use. Bilirubin will go to the liver to be incorporated into Bile, and Hemosiderin will go back to the bone marrow to serve as a source of iron for new, growing erythrocytes.

In Figure D, another Macrophage is seen as it engulfs a dying Neutrophil. The Neutrophil (NP), which is quite large, nearly fills the field. The only portions of the Macrophage visible here are part of its cell body and the pseudopodial extensions (PE) with which it embraces the Neutrophil.

Figure A. Fibroblasts in loose connective tissue. C, Collagen fibrils; G, Golgi Complex; N, Nucleus; Nu, Nucleolus; RER, rough Endoplasmic Reticulum; *, peripheral ring of Heterochromatin. 12,000 X Figure B. Mast Cell from the Lamina Propria of the human tongue. CT, connective tissue; G, electron-dense cytoplasmic granules; N, Nucleus of Mast Cell. 7,000 X Figure C. Macrophageengulfing Erythrocyte in monkey spleen. E, Erythrocyte; L, Lysosome; M, Macrophage; N, Nucleus; R, reticular fibers. 9,000 X Figure D. Macrophageengulfing Neutrophil in monkey spleen. M, Macrophage; NP, Neutrophil; PE, pseudopodial extension of Macrophage. 13,500 X