Connective and other mesenchymal tissues with their stains

Collagen fibers

These are the most frequently encountered of all the fibrous types of intercellular substance. They can occur as individual fibers, e.g. in loose areolar tissues where they are arranged in an open weave system, or as large bundles of fibers clumped together to form structures of great tensile strength, e.g. in tendon. Individual collagen fibers do not branch and when viewed by polarized light they are strongly birefringent, but lack dichroism.

Types of collagen

There are four major types of collagen (designated I–IV ), although several minor types are recognized. The production of the different types is under genetic control, each reflecting slight variations in the α-chain composition but all displaying the same characteristic amino acid content.

Type I

This forms the thick collagenous fibers which have been demonstrated histologically and form the bulk of the body’s collagen. It appears under the electron microscope as bundles of tightly packed thick fibrils, 75 nm diameter, with little interfibrillar substance. The fibrils show the characteristic 64 nm axial periodicity. The prominence of the ‘cross-banding’ in Type I collagen is thought to be due to the lack of interference from interfibrillar ground substance (Fig. 12.1) .

Type II

This is found in hyaline and elastic cartilage and is produced by chondroblasts. The fibers are thin and composed of fibrils arranged in a meshwork with copious amounts of proteoglycans. It is usually not visible by light microscopy. The Type II fibers found in articular cartilage are thicker and ultrastructurally resemble Type I fibers. The cross-banding of Type II collagen is less evident due to the masking effect of the abundant interfibrillar material.

Type III

This collagen, known as reticular fibers, is found only in those tissues which also contain type I collagen, e.g. lung, liver, spleen and kidney. Ultrastructurally, reticular fibers are loosely packed fibrils surrounded by abundant carbohydrate-rich interfibrillar material. The argyrophilia of reticular fibers is due to the proteoglycan content of the fibers and is not dependent upon the proteins of the fibrils themselves. Type III collagen provides a limited amount of support, but allows some motility and the easy diffusion and exchange of metabolites.

Type IV

This has been characterized in structures identified morphologically as basement membranes. It is generally accepted that it does not form fibers or fibrils visible with light microscopy. Electron microscopy reveals a random organization of fine fibrils forming a feltwork-like structure in all basement membranes. Type IV collagen is associated with large amounts of carbohydrate complexes explaining the strong reaction of basement membranes to the periodic acid-Schiff (PAS) method.

Types V and VI

These are similar. Type V collagen is produced in small quantities by a wide range of cells which include connective tissue, endothelial and some epithelial cells. It remains in close contact with the cell surface and is thought to be involved in the attachment of cells to adjacent structures and the maintenance of tissue integrity. Type VI collagen is a disulfide-rich variant which has been identified in boundary zones where interstitial collagenous fibers (types I and II) are linked to non-collagenous elements.

Type VII

This is the major structural component of anchoring fibrils which secure the basement membrane to the underlying dermis.

Reticular fibers

These are the fine delicate fibers which are found connected to the coarser and stronger collagenous type I fibers. They provide the bulk of the supporting framework of the more cellular organs, e.g. spleen, liver and lymph nodes, where they are arranged in a three dimensional mesh network providing a system of individual cell support ( Fig. 12.2 ). 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 but appear indistinct in H&E stained preparations. Reticular fibers may be demonstrated in paraffin sections using an argyrophil-type silver impregnation technique, or in frozen sections by the PAS 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 oxytalan, elaunin and elastic have a fibrillar, amorphous, or mixed structure respectively. They 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 their location from fine, single fibers as in the upper dermis to the membrane-like structures of the internal and external elastic laminae in the large arteries. In the latter, the elastic membranes are interrupted by minute holes called fenestrae (from the Latin ‘ fenestra’ for window). These fenestrae permit diffusion of materials through the otherwise impermeable membrane. High resolution electron microscopic examination has demonstrated that elastic fibers consist of two quite distinct components: the amorphous substance which biochemically is consistent with the protein elastin, and a second component which shows a periodicity of 4–13 nm and is microfibrillar in nature, the elastic fiber microfibrillar protein (EFMP).

Viewed in transverse section, the central core of the elastic fiber is composed of the amorphous protein elastin surrounded by a ring or band of elastin-associated microfibrils (EAMF). The proportions of the two components alter with the age of the fiber and probably the age of the subject. The dominant fraction in young fibers is the microfibrillar protein but in older fibers, the amorphous protein accounts for over 90%. The basic molecular unit of elastin is a linear polypeptide with a molecular weight of approximately 72 kilodaltons (kDa), ‘soluble 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. The polypeptides are transported out of the fibroblasts or smooth muscle cells and the cross-linking occurs in the extracellular spaces.

EFMP has an amino acid content which is quite distinct biochemically from elastin protein. It is particularly rich in amino acids which are lacking, or only present in small quantities in elastin. The content of cysteine in EFMP is high because of the presence of numerous disulfide linkages and this is significant for the staining properties of elastic fibers. A number of carbohydrate complexes, ‘structural glycoproteins’ ( ) are also associated with EFMP and significant in the staining of elastic fibers. Elastic fibers are acidophilic, congophilic and refractile. Following oxidation, they are 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 PAS reaction. They may be seen in routine H&E stained sections or by the use of a relatively simple stain e.g. the Taenzer-Unna orcein method, or the more lengthy and complex, e.g. Weigert resorcin-fuchsin method. Physical and biochemical changes are seen with increasing age in elastic fibers. These may include splitting and fragmentation, alteration of the ratio of EFMP to elastin and increases in the levels of calcium and glutamic and aspartic acids. These changes are readily visible in skin which becomes wrinkled and ‘loose-fitting’.

Basement membranes

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

• Lamina rara or lamina lucida: adjacent to the surface cells and composed mainly of carbohydrate complexes. It is apparently continuous with the glycocalyx of the surface cells and thought to be produced by the surface cells and not by the underlying connective tissue cells.

• Lamina densa or basal lamina: 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 ( ).

• Lamina reticularis: 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 but most are 15–50 nm thick. The kidney glomerular basement membrane (GBM) however is particularly thick, averaging 350 nm in a healthy adult. The ultrastructural appearance of the GBM also differs from that of other basement membranes, the central lamina densa is bordered on both sides by a lamina rara. These membranes are strongly positive with the PAS reaction and any other oxidation-aldehyde demonstration techniques, e.g. methenamine silver, Gridley and Bauer-Feulgen, as a result of their carbohydrate content. The basement membrane can also be stained by MSB or Azan trichrome methods.

Fibroblasts

This cell is responsible for the production of collagen fibers and probably the amorphous intercellular substance which binds the fibers together. Many authors refer to the young active secretory cell as the fibroblast, and reserve the term fibrocyte for the older non-secretory stage of development. The two stages may be distinguished under the microscope. The nucleus of the active spindle shaped fibroblast contains a prominent nucleolus and is surrounded by abundant, slightly basophilic cytoplasm. The thinner spindle shaped fibrocyte has an ovoid flattened nucleus with scanty chromatin, no nucleolus and difficult to distinguish cytoplasm. The fibroblasts are responsible for repair processes in the body and will accumulate at the edge of sites of injury and secrete fibrous intercellular substances which ultimately form scar tissue.

Fat cells or adipocytes

These are unique in the cells which differentiate from the mesenchymal cell as their main function is storage and not the production of intercellular substances or defense mechanisms. The first sign of development of a fat cell is the accumulation within its cytoplasm of tiny droplets of lipid material. These gradually increase in size until the cell loses its previous shape and appears as a swollen object with the nucleus and residual cytoplasm forced to one side.

Involuntary smooth muscle

This develops from mesenchymal cells which elongate themselves to form tapered cells of 20–50 μ m in length. It is found in the bowel, skin and bladder where it is involved in automatic peristalsis and reflex functions. The size of the muscle cell, or fiber, varies enormously depending upon the site where the cells are found. The cytoplasm contains bundles of myofilaments called myofibrils which are up to 1 mm in diameter. These are surrounded by the sarcoplasm, which constitutes the remainder of the cell. The centrally situated nuclei with their fine chromatin pattern are lightly stained with hematoxylin and have an elongated appearance which may contain one or two nucleoli. In cells which contract extensively and regularly, the nuclei may take on a ‘concertina’ shape which enables them to fit comfortably into the cell when it is in a state of full contraction.

Voluntary striated muscle

This makes up the bulk of the body’s musculature and is widely distributed over all parts of the skeleton, hence its alternative name of skeletal muscle. It is the type of muscle over which there is voluntary control. It is capable of briefly producing tremendous forces and also of maintaining a state of ‘semi-contraction’ for long periods of time, e.g. holding the body upright whilst standing. Certain functions, e.g. respiration, become almost automatic and driven by the nervous system although voluntary control is maintained. Like smooth muscle, it is composed of elongated eosinophilic cells, but in striated muscle these are much larger and longer, up to 40 mm in length and 40 μm in diameter. The cells themselves do not taper towards the ends but are more cylindrical. The nuclei are elongated and stain with hematoxylin. There is often more than one nucleus present in each cell and these are situated peripherally and often in contact with the sarcolemma or cell membrane.

The most obvious and striking characteristic of striated muscle is the striations or stripes which cross the cells at right angles to their long axes. On closer examination, and by polarized light and electron microscopy, the striations can be seen to be alternating light and dark bands or discs. The darker bands are birefringent and referred to as A-bands or Q-bands, the paler bands are isotropic and known as I-bands. Occasionally there is a narrow, dark band, the Z-line, running through the center of the paler I-bands. Studies of relaxed voluntary muscle with the electron microscope have shown the presence of a fourth extremely thin band, the H-line or H-disc, running through the A-band ( Fig. 12.3 ). With the aid of the electron microscope it may be noted that these numerous discs or bands are not complete structures crossing the muscle cells, but are composed of striated myofibrils. The organized arrangement of these striae gives the appearance of discs. There is a well-developed system of mitochondria and the sarcoplasmic reticulum between these myofibrils. Stored in the sarcoplasm, adjacent to the sarcoplasmic reticulum, is an abundant store of glycogen to provide an immediate source of energy.

Striated cardiac muscle

This is only found in the heart where it constitutes the bulk of the myocardium and differs from smooth and striated muscle in several ways. It is not composed of distinctly separate cells but of cells which branch and anastomose frequently. The cytoplasm contains myofibrils and sarcoplasm and exhibits a ‘striated’ appearance similar to that of voluntary muscle. The striations are less distinct as the myofibrils are not arranged to coincide as regularly as in voluntary striated muscle.

One feature unique to cardiac muscle is the presence of intercalated discs. These were originally believed to be another type of striation or stripe but with the electron microscope it can be seen that these discs represent the end to end junction of adjacent cardiac muscle cells. The nuclei are placed centrally in the cells and stain lightly with hematoxylin. Situated in the intercellular spaces created between the anastomosing of the cells are the blood vessels, lymphatics and nerves to maintain and nourish the cardiac muscle.

General structure of muscle

All the different types of muscle contain considerable amounts of connective tissue. Each complete muscle is surrounded by an envelope of collagen and elastic fibers known as the ‘epimysium’. There are numerous bands or sheets of connective tissue arising from the epimysium which divide the whole muscle into bundles of muscle cells, the ‘perimysium’. A connective tissue sheath, the ‘endomysium’ covers each individual muscle cell. This complex system of interconnecting collagen fibers is continuous with the attachment points of the muscle, the fine sheets of collagen fibers give way to broader, stronger bands of dense connective tissue which continue to form the tendon.