Connective and mesenchymal tissues with their stains

11 Connective and mesenchymal tissues with their stains


Connective tissue is one of the four tissue types found throughout the body. The term ‘connect’ comes from the Latin word ‘connectere’ meaning ‘to bind’. Its main function is to connect together and provide support to other tissues of the body. During embryonic development, the ectoderm and endoderm are divided by a germ layer called the mesoderm, generically known as mesenchyme. This comes from the Greek words ‘mesos’ meaning middle and ‘enchyma’ meaning infusion. It is from the mesenchyme that the connective tissues develop.

In almost any type of connective tissue there are three elements: the cells, the fibers, and the amorphous ground substance. The identification of the cells may be based upon their appearance in areolar and loose tissues that can be considered the main ‘packing’ material in the adult. This can also be considered as a prototype of the connective tissue. The connective tissues are divided into the following groups:

Connective tissue usually consists of a cellular portion in a surrounding framework of a non-cellular substance. The ratio of cells to intercellular substance varies from one type of connective tissue to another, as does the primary function of the connective tissue. For example, bone has only a few cells in a usually dense, rigid intercellular substance, with its main function being that of providing strength and support. The cell types of connective tissue can include entities such as fibroblasts, mast cells, histiocytes, adipose cells, reticular cells, osteoblasts and osteocytes, chondroblasts and chondrocytes, blood cells, and blood-forming cells. Some connective tissue has little substance and consists primarily of cells whose functions are not those of production of intercellular substance, such as adipose tissue. Since they do not connect or support the other body tissues they are included under connective tissues as they probably derive from the same parent cell. The parent cell of the entire series is the embryonic mesenchyme cell, which is rarely found in adults.

The intercellular substance is usually composed of both amorphous (non-sulfated and sulfated mucopolysaccharides) and formed elements (collagen, reticular fibers and elastic fibers). These are the non-living parts of the connective tissues. The nature of the intercellular substance varies according to its function. It may be extremely hard and dense, in cortical bone, or soft as in umbilical cord. Its microscopic appearances are also variable, some being fibrillar, whereas others are completely homogeneous. Intercellular substances may be readily classified into two main groups by their microscopic appearance:

Formed or fibrous intercellular substances

A frequent fault among histologists is to speak of collagen, reticulin, and elastin, when in reality they mean collagenic fibers, reticular fibers, and elastic fibers. The former terms relate to the protein compound that is predominant in the particular fiber and should not be used to describe the connective tissue fiber itself.

Collagenic fibers

These are the most frequently encountered of all the fibrous types of intercellular substance, and are found in large quantities in most sites in the body. They may occur as individual fibers, as in loose areolar tissues, arranged in an open weave system, or as large bundles of fibers clumped together to form structures of great tensile strength, such as tendon. Individual collagenic fibers are never seen to branch, although some bundles of fibers do branch frequently. When viewed by polarized light, they are strongly birefringent, but lack dichroism.

Types of collagen

Four major types of collagen and several minor types have been characterized and described. The production of the different types is under genetic control, each reflecting slight variations in the α-chain composition. They all display the same characteristic amino acid content.

Reticular fibers

These are the fine delicate fibers that are found connected to the coarser and stronger collagenous fibers (Type I fibers). They provide the bulk of the supporting framework of the more cellular organs (e.g. spleen, liver, lymph nodes, etc.), where they are arranged in a three-dimensional network to provide a system of individual cell support (Fig. 11.2). On light microscopic examination, reticular fibers are weakly birefringent, the weak reaction being attributed to their lack of physical size and the masking effect of the interfibrillar substance. They are seen to branch frequently and appear indistinct in H&E-stained preparations. The characteristics of reticulin fibers in human kidney cortex have been studied using immunohistochemical means. Antibodies directed against Type I and Type III collagens, their corresponding amino peptides and decorin (PG-II) revealed that in this organ the reticulin fibrils consist of hybrids of Type I and Type III collagens. Double immuno-electron microscopy shows that 20–25 nm fibrils consist mainly of Type I collagen, whereas the larger fibrils, 30–35 nm, label simultaneously for Type I and Type III collagens. Most fibrils larger than 40 nm in diameter label for Type III collagen. Reticular fibers may be demonstrated distinctly, in paraffin sections, using one of the many argyrophil-type silver impregnation techniques available or, in frozen section, by the periodic acid-Schiff technique. Both methods of demonstration are dependent upon the reactive groups present in the carbohydrate matrix, and not upon the fibrillar elements of the fiber.

Elastic fibers

The elastic system fibers (i.e. oxytalan, elaunin, and elastic fibers) have, respectively, a fibrillar, amorphous, or mixed structure. The elastic fibers may be found throughout the body but are especially associated with the respiratory, circulatory, and integumentary systems. Their appearances under the light microscope may vary considerably according to location, from fine, single fibers, as in the upper dermis, to membrane-like structures (‘internal and external elastic laminae’) in the large arteries. In the latter situation, the elastic membranes are interrupted by minute holes called fenestrae (Latin ‘fenestra’ – window) which permit diffusion of materials through the otherwise impermeable membrane. Recent high-resolution electron microscopic examination has demonstrated that elastic fibers consist of two quite distinct components. There is an amorphous substance which, biochemically, is consistent with the protein elastin, and also a second component, which shows a periodicity of 4–13 nm, that is microfibrillar in nature, and has been termed elastic fiber microfibrillar protein (EFMP). These micro-fibrils, sometimes also called elastin-associated microfibrils (EAMF), are ubiquitous connective tissue structures that are believed to provide tensile strength and flexibility to numerous tissues. They may also act as a scaffold for elastin deposition.

When viewed in transverse section, the central core of the elastic fiber is seen to be composed of the amorphous protein elastin, surrounded by a ring or band of EFMP. The proportions of the two components seem to alter with the age of the fiber (and probably also with the age of the subject). In young fibers, the dominant fraction is the microfibrillar protein. In older fibers, the amorphous protein accounts for over 90% of the fiber content. The basic molecular unit of elastin is a linear polypeptide with a molecular weight of approximately 72 kilodaltons (kDa). This subunit has been referred to as ‘soluble elastin’ or ‘tropoelastin’. One of the characteristic features of elastic fibers is the presence of cross-linking which binds the polypeptide chains into a fiber network. Desmosine and its isomer, isodesmosine, are the cross-linking compounds involved. The polypeptides are transported out of the fibroblasts or smooth muscle cells and the cross-linking occurs in the extracellular spaces.

Elastic fiber microfibrillar protein has an amino acid content that is quite distinct, biochemically, from that of elastin protein. It is particularly rich in amino acids, which are lacking or present in only small quantities in elastin. The content of cysteine in EFMP is high, reflecting the presence of numerous disulfide linkages that will be of significance when the staining properties of elastic fibers are considered later. Associated with EFMP are a number of carbohydrate complexes, termed ‘structural glycoproteins’ (Cleary & Gibson 1983); the significance of these in the staining of elastic fibers will also be considered later. For a more detailed account of elastic fiber composition and biochemistry, reference should be made to the work of Cleary and Gibson (1983), Uitto (1979), or Bailey (1978). Elastic fibers are acidophilic, congophilic, and refractile. Following oxidation, they are quite strongly basophilic due to the formation of sulfonic acid groups from the disulfide linkages of the EFMP. Young fibers with a high content of EFMP show a positive periodic acid-Schiff reaction. They may be seen in routine H&E- stained sections, but, for exacting studies, numerous more selective techniques are available. These may be relatively simple, e.g. the Taenzer-Unna orcein method, or more lengthy and complex, e.g. Weigert resorcin-fuchsin methods. With increasing age of the elastic fibers, physical and biochemical changes are seen to occur. These may include splitting and fragmentation, alteration of the ratio of EFMP to elastin, and increases in the levels of glutamic and aspartic acids and calcium. These changes are readily visible in the skin of the subject, which becomes wrinkled and ‘loose-fitting’. A more serious problem occurs with the loss of elasticity of the elastic arteries.

Oxytalan fibers

Oxytalan fibers were first described by Fullmer and Lillie (1958) in periodontal membranes. More recently they have been demonstrated in a wide variety of tissues, both normal and abnormal (Alexander & Garner 1977; Cleary & Gibson 1983; Goldfischer et al. 1983). On light microscopic examination, oxytalan fibers may be distinguished from mature elastic fibers by their failure to stain with aldehyde fuchsin solutions, unless they have been previously oxidized by potassium permanganate, performic acid, or peracetic acid. They have also been reported to remain unstained following Verhöeff’s hematoxylin, with or without prior oxidation. Following electron microscopic examination by a number of workers, it has been suggested that oxytalan fibers are similar to, if not identical to, EFMP fibers. They appear to be composed of microfibrillar units, 7–20 nm in diameter, with a periodicity of 12–17 nm. Their periodicity is made more conspicuous by pretreatment with ruthenium red. From their morphology, localization, and staining properties, it seems possible that oxytalan fibers may represent an immature form of elastic tissue. It has also been suggested by Goldfischer et al. (1983) that microfibrils and oxytalan fibers may have a role beyond that of elastogenesis and may involve ‘anchoring’ mechanisms between collagen fibers, stromal cells, lymphatic capillary walls, mature elastic fibers, muscle cells, etc.

Basement membranes

Basement membranes are found throughout the body as a resilient matrix, separating connective tissues from epithelial, endothelial or mesothelial cells, muscle cells, fat cells, and nervous tissues. They support the epithelial cells of mucosal surfaces, glands, and several other structures, for example renal tubules. They also support the endothelial cells lining blood vessels, capillaries, etc. The basement membrane is not homogeneous, but is divided into three zones or layers:

The lamina rara (lucida) is adjacent to the surface cells and is composed mainly of carbohydrate complexes. This layer is apparently continuous with the glycocalyx of the surface cells and it has been suggested that the lamina rara is produced by the surface cells and not by the underlying connective tissue cells. The lamina densa is composed of a feltwork of microfibrils which have been immunohistochemically identified as predominantly Type IV collagen with a lesser amount of Type V collagen. Type IV collagen is associated with relatively large amounts of structural glycoproteins, mainly laminin and fibronectin, and small amounts of proteoglycans, principally heparan sulfate (Junqueira & Montes 1983; Laurie & Leblond 1983). The lamina reticularis is seen as a layer containing fibrous elements, which are continuous with the underlying connective tissue fibers.

The thickness of the basement membrane varies from site to site; most are in the range 15–50 nm. The glomerular basement membrane (GBM) is particularly thick, up to 350 nm in a healthy adult. The ultrastructural appearance of the GBM also differs from that of other basement membranes, in that the central lamina densa is bordered on both sides by a lamina rara. The sequence of the ultrastructural elements in the GBM is from the capillary lumen outwards: endothelial cell, endothelial-associated lamina rara, lamina densa, epithelial-associated lamina rara, epithelial cell (podocyte). In H&E-stained sections of most tissues, basement membranes are difficult to distinguish; in the glomerulus, they are more conspicuous, particularly in disorders such as membranous nephropathy or diabetes, where they can be markedly thickened. For more critical examination, a number of techniques are available. As a result of their carbohydrate content, the membranes are strongly positive by the periodic acid-Schiff reaction and by any other oxidation-aldehyde demonstration techniques, e.g. methenamine silver, Gridley, Bauer-Feulgen, etc. In sections by the MSB or Azan trichrome methods, the basement membrane stains intensely by the larger molecule, acid dye.

Methenamine silver microwave method

This method delineates the glomerular basement membranes. Methenamine silver demonstrates the carbohydrate component of basement membranes by oxidizing the carbohydrates to aldehydes. Silver ions from the methenamine-silver complex are first bound to carbohydrate components of the basement membrane and then reduced to visible metallic silver by the aldehyde groups. Toning is with gold chloride, and any unreduced silver is removed by sodium thiosulfate. The use of a microwave oven is recommended for the method and the technique should be followed exactly for optimal results. The method below is for five slides. Note: if you do not have five slides, then include blank slides but do not use more than five.

Periodic acid-methenamine silver microwave method for basement membranes (Brinn 1983; Carson 1997)

Connective tissue cells

Connective tissues consist of a non-living framework in which cells function and live. The cellular component is an important aspect of this group of tissues. The parent cell of the entire series of connective tissues is the undifferentiated mesenchymal cell. From this develop many varied cells, each with its different function.

Connective tissues

The physical characteristics of the cells and the inter-cellular substances vary considerably and they may be divided into groups, according to the ratio of cells to intercellular substance, and the types of cells and intercellular substance:


The connective tissues discussed previously possess great tensile strength (that is, will resist diverging forces) but, when placed under pressure, they will bend. The structural characteristics of cartilage partially overcomes this problem. It consists of a fairly dense network of collagenic fibers encased in, or bonded with, an amorphous intercellular substance of chondroitin sulfate, which is in the form of a thin gel. Cartilage is distributed throughout the body in sites where the functions it is required to perform are slightly different. Hence, although it has an almost ‘standard’ form known as hyaline cartilage, it does have two other modified forms, elastocartilage and fibrocartilage. These will be considered later.

Microscopically, hyaline cartilage is composed of a matrix of apparently homogeneous intercellular substance, and is fibrillar in structure, containing large numbers of collagenic fibers. In the matrix are the cellular components of cartilage, the chondrocytes, which reside in spaces in the matrix known as ‘lacunae’. There may be one cell or as many as six cells in each lacuna. In a fresh state the chondrocytes will fill the lacunae, whereas in stained sections they will often appear rather shrunken. The cytoplasm contains glycogen and lipid, and the nuclei are spherical with one or more nucleoli. Young immature chondrocytes tend to be rather small and flattened. As they mature they become larger and more rounded. Immediately surrounding the cell lining the lacuna is what appears to be an intercellular substance of a different type from that which comprises the bulk of the matrix.

Hyaline cartilage is the most common cartilage and is found in the larynx, the bronchus, the nose and as the articulatory surface of joints. When thoroughly lubricated with synovial fluid, the surface of articular cartilage will take on the appearance of a high polish and is ideally suited for the bearing surfaces of joints. Although hyaline cartilage is slightly elastic, in some instances this is not adequate. Elastocartilage is found where more elasticity is required. It contains as many collagenic fibers but has the addition of elastic fibers. It is found in the external ear and the epiglottis.

Fibrocartilage is found in sites such as tendon inserts where the tensile strength of hyaline cartilage is insufficient. The collagenic fibers of hyaline cartilage are arranged with no regular pattern; in fibrocartilage they are packed in rows parallel to the direction of the force. Between the collagenic fiber bundles lie fibroblasts and rows of chondrocytes and intercellular substance. Cartilage develops from the mesenchymal cells that differentiate into chondroblasts and lay down intercellular substance. They mature into chondrocytes and the cartilage in this form can live for long periods, as it does in joints. Mature, hypertrophied chondrocytes will produce alkaline phosphatase, which brings about a reaction whereby insoluble calcium salts are precipitated in the matrix of the cartilage. As the calcification proceeds, the nutriments needed by the chondrocytes are cut off and the cells use up their stored glycogen and die. The calcified cartilage is not a permanent formation as it has no living cellular component, and it soon breaks down and the tissue loses all its supporting structures.

Muscular tissue

The purpose of muscular tissue is to provide the power to enable the body to move and to function. Although muscular tissues are readily divided into three distinct categories, all types are composed of similar constituents and their mode of providing power and movement is also similar. Muscles provide power and movement by contracting their cells, so shortening their overall length, and thus pulling closer together the points to which the muscle is attached. Many cells in the body share this ability to contract and change shape. This phenomenon is due to the presence of three proteins and the interactions between them:

α-Actin forms a base, which allows a strand of actin to become attached. Myosin in turn attaches to this actin strand and is able to move up the strands towards the base by means of a ratchet-like mechanism. Because myosin is double-headed, it interacts simultaneously with two separate actin strands. The movement up the strands from these different base plates pulls the strands together, causing the fiber to contract. A single muscle fiber of skeletal muscle is long and thin and comprises many myofibrils. These subcellular components run parallel to the fiber’s long axis. Each myofibril is built up of a large number of identical contractile units called sarcomeres. On full contraction this can shorten by about 30% of its resting length. The three types into which muscular tissues may be classified are:

Dec 13, 2017 | Posted by in HISTOLOGY | Comments Off on Connective and mesenchymal tissues with their stains
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