Connective Tissue

Connective Tissue

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

Extracellular Substance

Collagen fibres

Collagen fibres are the dominant fibre type in most connective tissues. The primary function of collagen fibres is to add strength to the connective tissue.
The thickness of the fibres varies from ~ 1 to 10 µm. Longitudinal striations may be visible in thicker fibres. These striations reveal that 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.
Microfibrils are assemblies of tropocollagen, which, in turn, is an spiral-like assembly of three collagen molecules (triple helix). The organisation of the tropocollagen within the microfibrils is highly regular. A small gap (60 nm wide) is found between the subsequent tropocollagens which form the microfibrils. Staining solutions used in electron microscopy tend to fill in these gaps, and the alignment of the gaps gives the microfibrils a cross-striated appearance (with 68 nm intervals)in EM images.
Coarse collagen fibres are formed by type I tropocollagen.

There are many different tropocollagen types around (currently named type I to XXI). These types differ in their content of the amino acids hydroxyproline and hydroxylysine. They also differ in the amount of carbohydrates attached to the collagen molecules. The different types of tropocollagen give the fibres the structural and functional features which are appropriate for the organ in which the fibres are found. Types I, II and III are the major fibre-forming tropocollagens. Tropocollagen type IV is an important structural component of the basal lamina.

A tensile force of several hundred kg/cm² is necessary to tear human collagen fibres. The fibres stretch by only 15-20%.

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 - coarse collagen fibres appear reddish brown in the same type of preparation.
Because of their different staining characteristics, reticular fibres were initially thought to be completely different from collagen fibres. Cross-striations with the same periodicity as in coarse collagen fibres are however visible using electron microscopy. We now know that reticular fibres consist of collagen - although the main type of tropocollagen found in reticular fibres, type III, is different from that of the coarse collagen fibres.
Reticular fibres give support to individual cells, for example, in muscle and adipose tissue.

Suitable Slides

sections of liver, spleen or lymph nodes - reticulin

Liver - Reticulin Stain The liver is one of the organs in which the cells are supported by a network of reticular fibres. They appear as fine black lines in this silver stained preparation. The fibres surround the individual sheets of liver cells (hepatocytes) and are the only fibrous connective tissue component supporting the cells. While providing support, the fine, open meshwork of reticular fibres facilitates the exchange of substances between the hepatocytes and the blood, which circulates in the irregularly shaped blood vessels (sinusoids) between the hepatocytes. Reticular fibres are also present in the connective tissue surrounding the larger vessels, which penetrate the parenchyma of the liver. Draw reticular fibres as they surround a nice piece of a row of liver cells at high magnification - include a suitable scale and label your drawing. Blood will not be visible in some types of preparations and the sinusoids appear empty.

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Elastic fibers

Elastic fibres are coloured in fresh tissues - they are light yellow - but this colouration is only visible if large amounts of elastic fibres are present in the tissue, for example, in the elastic ligaments of the vertebral column. Special stains are necessary to show elastic fibres in tissue sections.
Resorcin fuchsin is one of these stains, which gives the elastic fibres a dark violet colour.
Light microscopy does not reveal any substructure in the elastic fibres. Electron microscopy shows that elastic fibres consist of individual microfibrils, which are embedded in an amorphous matrix. The matrix accounts for about 90% of the fibre and is composed of the protein elastin. Neither the elastin nor the microfibrils are collagens.
Elastic fibres can be stretched to about 150% of their original length. They resume their original length if the tensile forces applied to the elastic fibres are relaxed.

Elastin is a somewhat odd protein in that its amino acid sequence does not determine a specific three-dimensional structure of the molecule. Instead, elastin remains unfolded as a "random coil". Elastin molecules are cross-linked to each other by desmosin and isodesmosin links, which are only found between elastin molecules. Tensile forces straighten the cross-linked mesh of elastin coils.

Suitable Slides

sections of blood vessels or skin - elastin

Skin, human - elastin & van Gieson or 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.
Collagen and elastic fibres intermingle in the dermis, i.e. the connective tissue beneath the epithelium of the skin. Immediately beneath the epithelium both fibre types are relatively fine - they appear much thicker in the deeper parts of the dermis. At least the internal elastic lamina should be visible in the smaller arteries which course through the dermis.
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.
Identify the artery and the vein in the section. Their walls contain large amounts of elastic fibres.
Blood vessels: draw a small section of the wall of a vessel, preferably an artery, at high magnification. Identify elastic laminae, fine and coarse elastic fibres in your drawing.
Skin: draw a small section of the dermis - preferablyof a part of the dermis where both the very fine and the coarse fibres are visible.