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School of Anatomy and Human Biology - The University of Western Australia

     Blue Histology - Connective Tissues

Topics

Lab Guides and Images

Extracellular Substance

Reticular Fibres - liver, reticulin

Elastic Fibres - artery, elastin & eosin

Connective Tissue Cells

Macrophages - liver, carbon injected, trichrome

Mast Cells - tongue, toluidine blue

Connective Tissue Types

Dense Regular Connective Tissue - tendon, van Gieson

Dense Irregular Connective Tissue - non-lactating breast, H&E

Adipose Tissue - kidney, trichrome and thick skin, H&E

Mesenchymal Connective Tissue - umbilical cord, H&E

Additional Resources

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Large Images
Search the Large Images page with these keywords: dense irregular connective tissue, dense regular connective tissue, reticular connective tissue, mesenchymal connective tissue, white adipose tissue, brown adipose tissue, macrophage, adipocyte, mast cell or elastic fibres.
VScope
Magnification & Stage Simulations
dense irregular connective tissue, human, H&E - non-lactating breast
reticular connective tissue, reticulin - liver
Focus & Stage Simulations
spleen, reticulin - reticular connective tissue
Self Assessment
Choose subject area "connective tissue" on the Quiz page.

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.


Elastic fibres

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. Which one contains more elastic fibres - artery or vein?

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.


 
Ground substance

Ground substance is found in all cavities and clefts between the fibres and cells of connective tissues. Water, salts and other low molecular substances are contained within the ground substance, but its main structural constituent are proteoglycans.
Ground substance is soluble in most of the solvents used to prepare histological sections and therefore not visible in ordinary sections.

Proteoglycans are responsible for the highly viscous character of the ground substance. Proteoglycans consist of proteins (~5%) and polysaccharide chains (~95%), which are covalently linked to each other. The polysaccharide chains belong to one of the five types of glycosaminoglycans, which form the bulk of the polysaccharides in the ground substance.

Hyaluronan (or hyaluronic acid) is the dominant glycosaminoglycan in connective tissues. The molecular weight (MW) of hyaluronic acid is very high (~ MW 1,000,000 ). With a length of about 2.5 µm hyaluronan is very large. Hyaluronan serves as a "backbone" for the assembly of other glycosaminoglycans in connective and skeletal tissue, which results in even larger molecule complexes (MW 30,000,000 - 200,000,000).
Hyaluronan is also a major component of the synovial fluid, which fills joint cavities, and the vitreous body of the eye.

The remaining four major glycosaminoglycans are chondroitin sulfate, dermatan sulfate, keratan sulfate and heparan sulfate. These glycosaminoglycans attach via core- and link-proteins to a backbone formed by the hyaluronic acid. The coiled arrangement of the hyaluronan and other attached glucosaminoglycans fills a roughly spherical space with a diameter of ~0.5 µm. This space is called a domain. Neighbouring domains overlap and form a more or less continuous three-dimensional molecular sieve in the interstitial spaces of the connective tissues.

The large polyanionic carbohydrates of the glycosaminoglycans bind large amounts of water and cations. The bound water in the domains forms a medium for the diffusion of substances of low molecular weight such as gases, ions and small molecules, which can take the shortest route, for example, from capillaries to connective tissue cells. Large molecules are excluded from the domains and have to find their way through the spaces between domains.

The restricted motility of larger molecules in the extracellular space inhibits the spread of microorganisms through the extracellular space. A typical bacterium ( 0.5 x 1 µm) is essentially immobilised in the meshwork formed by the domains. The pathogenicity of a bacterium is indeed to some extent determined by its ability to find its way through the mesh, and some of the more invasive types produce the enzyme hyaluronidase, which depolymerises hyaluronic acid.

The components of the ground substance, collagen, elastic and reticular fibres are synthesised by cells of the connective tissues, the fibrocytes.

 
Connective Tissue Cells

Connective tissue cells are usually divided into two groups based on their ability to move within the connective tissue. Fibrocytes (or fibroblasts) and fat cells are fixed cells. Macrophages, monocytes, lymphocytes, plasma cells, eosinophils and mast cells are wandering cells.

Fibrocytes

Fibrocytes are the most common cell type in connective tissues. They are the "true" connective tissue cells. Usually only their oval, sometimes flattened nuclei are visible in LM sections. The cytoplasm of a resting (i.e. inactive) fibrocyte does not contain many organelles. This situation changes if the fibrocytes are stimulated, for example, by damage to the surrounding tissue. In this case the fibrocyte is transformed into a fibroblast, which contains large amounts of the organelles which are necessary for the synthesis and excretion of proteins needed to repair the tissue damage (Which ones?). Fibrocytes do not usually leave the connective tissue. They are, however, able to perform amoeboid movement.

The terms fibrocyte and fibroblast refer here to the inactive and active cells respectively - at times you will see the two terms used as synonyms without regard for the state of activity of the cell.

Reticular cells

Reticular cells are usually larger than an average fibrocyte. They are the "fibrocytes" of reticular connective tissue and form a network of reticular fibres, for example, in the lymphoid organs. Their nuclei are typically large and lightly stained (H&E) and the cytoplasm may be visible amongst the cells which are housed within the network of reticular fibres.

Adipocytes

Fat cells or adipocytes are fixed cells in loose connective tissue. Their main function is (what surprise!) the storage of lipids. If "well fed" the cytoplasm only forms a very narrow rim around a large central lipid droplet. The flattened nucleus may be found in a slightly thickened part of this cytoplasmic rim - if it is present in the section, which may not be the case since the diameter of an adipocyte (up to 100 µm) is considerable larger than the thickness of typical histological sections. A "starving" adipocyte may contain multiple small lipid droplets and gradually comes to resemble a fibrocyte.

Lipid storage/mobilisation is under nervous (sympathetic) and hormonal (insulin) control. Adipocytes also have an endocrine function - they secrete the protein leptin which provides brain centers which regulate appetite with feedback about the bodies fat reserves.
Leptin deficiency in experimental animals results in obesity.

Adipocytes are very long-lived cells. Their number is determined by the number of preadipocytes (or lipoblast) generated during foetal and early postnatal development.

Macrophages

Macrophages arise from precursor cells called monocytes. Monocytes originate in the bone marrow from where they are released into the blood stream. They are actively mobile and leave the blood stream to enter connective tissues, where they differentiate into macrophages. Macrophages change their appearance depending on the demand for phagocytotic activity. Resting macrophages may be as numerous as fibrocytes.
Resting macrophages are difficult to distinguish from fibrocytes in H&E stained sections.


 
Suitable Slides
sections of liver or lymph nodes - carbon injected animal - trichrome or H&E
 
sections of lung - H&E
In the lung, macrophages "patrol" the respiratory surfaces and ingest airborne particles which settle there. They "retire" to the connective tissue of the lung were they can be identified by the accumulations of fine particles in their cytoplasm.
 
sections containing irregular connective connective tissues - Alcian blue & van Gieson
In Alcian blue & van Gieson stained sections macrophages may appear as intensely green, roundish cells with a dark nucleus.

Liver, rabbit - ink injected, trichrome
Macrophages are usually difficult to distinguish from other cell types in connective tissues. One way to visualize them is to inject an experimental animal with very fine carbon particles. Macrophages which come into contact with the circulating particles will phagocytose some of them. In sections the particles will be visible as dark, black-brown accumulations in the cytoplasm of the macrophages.
Draw a few macrophages in situ.

Macrophages found in the liver are also called Kupffer cells. They adhere to the epithelial lining of the liver sinusoids, i.e. blood filled spaces between the liver cells. Blood will not be visible in some types of preparations and the sinusoids appear empty.

Once you have identified macrophages, go hunting for some good collagen - in this trichrome stains the collagen fibres will appear green(ish). Typically you will see them only in the connective tissue surrounding larger blood vessels. Improve your knowledge on epithelia and look out for ducts lined by a simple cuboidal or columnar epithelium. What is flowing in these ducts?


Mast cells

Mast cells are - like macrophages, lymphocytes and eosinophils - in demand when something goes wrong in the connective tissue. Quite a few of them are present in healthy connective tissue as they stand on guard and monitor the local situation. The cytoplasm of mast cells is filled by numerous large vesicles. Mast cells discharge the contents of these vesicles if they come in contact with antigens, for example, proteins on the surface of an invading bacterium or, in allergic reactions, in response to antigens found, for example, on the surface of pollen grains.

The most prominent substances contained in the vesicles are heparin and histamine. They increase blood flow in close by vessels and the permeability of the vessel walls to plasma constituents and other white blood cells. By facilitating access to the area, mast cells facilitate an immune response to the antigen which triggered the release histamine and heparin.

Other connective tissue cells

Lymphocytes and plasma cells

Lymphocytes are usually small cells (6 - 8 µm). Their nuclei are round and stain very dark. The cytoplasm forms a narrow rim around the nucleus and may be difficult to see. There are many of them in the connective tissue underlying the epithelia of the gastrointestinal tract but usually much fewer in other connective tissues. Again, this situation may change - in this case with immunological reactions. Some lymphocytes may differentiate into plasma cells. Plasma cells are lymphocytes which produce antibodies. To accommodate the necessary organelles for this function the size of the cytoplasm increases dramatically and the cells become basophilic. Plasma cells can occasionally be spotted in the loose connective tissue present in sections.
Like eosinophilic cells and monocytes, lymphocytes are white blood cells. More information about these cell types can be found on the Blood page.

Eosinophilic cells

Eosinophilic cells are typically rounded or oval, large cells, which contain large amounts of bright red granules in their cytoplasm. They originate, like the monocytes, in the bone marrow. They enter connective tissues early in inflammatory reactions, where they phagocytose antigen-antibody complexes. Their numbers in healthy connective tissue vary with location, but a few of them can usually be found.

Mesenchymal cells

During development, mesenchymal cells give rise to other cell types of the connective tissue. A small number of them may persist into adulthood. Mesenchymal cells are smaller than fibrocytes and difficult to detect in histological sections. They may regenerate blood vessels or smooth muscle which have been lost as a consequence of tissue damage.


 
Suitable Slides
sections of  tongue, skin, mesentery or other sections containing epithelia and / or loose connective tissue - toluidine blue, cresyl violet

Mast Cells, Tongue - toluidine blue and
Mesentry, Rat  - cresyl violet
Mast cells are relatively frequent in the connective tissue benath the epithelium and between the muscle fibres of the tongue. In most connective tissue cells and the muscle fibres only the nucleus is stained by the toluidine blue. The cytoplasm of the mast cells is however filled with dark, blue / violet grains which represent their secretory vesicles. At low magnification mast cells stand out as large, dark dots among smaller and lighter stained nuclei and among the very weakly stained remaining connective tissue components.
Draw a few mast cells in situ and label both the mast cells and some of the surrounding tissue components.


 
Connective Tissue Types

Loose connective tissue and dense connective tissues

These two tissues are distinguished according to the relative amounts of fibres they contain. Dense connective tissues are completely dominated by fibres. They are subdivided according to the spatial arrangement of the fibres in the tissue.

In dense irregular connective tissue the fibres do not show a clear orientation within the tissue but instead form a densely woven three-dimensional network. A good example is the dermis of the skin.

We talk about regular dense connective tissue if the fibres run parallel to each other. Good examples of regular dense connective tissue are tendons, ligaments and the fasciae and aponeuroses of muscles.

Loose connective tissue is relatively cell rich, soft and compliant. It is also rich in vessels and nerves. It is best understood as a kind of generalised connective tissue in which all connective tissue cell types may occur. Loose connective tissue may occur in some special variants: mucous connective tissue, reticular connective tissue and adipose tissue.


 
Suitable Slides
sections of tendons or ligaments - van Gieson, H&E

Muscle-Tendon Junction, rat - van Gieson
In van Gieson stained preparations collagen stains dark red while other tissue components appear in varying shades of grey (nuclei) and yellow (cytoplasm). Areas of dense regular connective tissue are usually easy to identify in these preparations. Coarse collagen fibres are aligned with each other with only very narrow opens spaces between them. Like in most other connective tissues, there will be only a few cells between the fibres. Their cytoplasm is difficult to identify but the nuclei can be seen scattered among the collagen fibres. Nuclei are often elongated, and their long axis runs parallel to the course of the collagen fibres
Sketch part of the regular dense connective tissue.

 
Suitable Slides
sections of skin or non-lactating mammary gland - H&E, van Gieson, trichrome

Non-lactating Breast - H&E
Dense irregular connective tissue forms the dermis of the skin, i.e. the layer of connective tissue immediately below the epithelium lining the surface of the skin. Beneath the skin forming the mammae (nipples), dense connective tissue areas are very extensive. This tissue surrounds the resting mammary gland. Both the high density of collagen fibres and the their irregular distribution are easily visible. Again, only a very small fraction of the tissue is taken up by cells. Like in van Gieson stained preparations, their cytoplasm is often not visible in H&E stained sections. Dark spots scattered between the collagen fibres represent the nuclei of the cells.
Draw part of the connective tissue including some fibrocytes.

The excretory ducts of the mammary glands are called lactiferous ducts. They are lined by a quite nice stratified columnar epithelium. If you are working with a section of non-lactating breast look for the lactiferous ducts in the connective tissue.


 
Reticular connective tissue

Reticular connective tissue consists of reticular cells and the network of reticular fibres formed by them. Most connective tissues contain reticular fibres, but only in reticular connective tissue are they the dominant fibre type. In a number of tissues and organs, reticular connective tissue forms the structural framework in which the cells of the organ are suspended. The open meshwork of fine fibres is particularly useful in tissues and organs in which diffusion and / or cell movements are functionally important, for example, in the liver, lymph nodes and the spleen.

 
Adipose tissue

Adipose tissue is essentially loose connective tissue containing large numbers of adipocytes. There are two types of adipose tissue, which derive their names from the colour of the tissue (white or brown) and the number of lipid droplets found in the adipocytes.

White adipose tissue does not only function in the storage of lipids. For example, in the palms of the hands, on the plantar surface (sole) of the feet and in the gluteal region (buttocks) it has a structural, cushioning function. In these regions, accumulations of adipocytes are surrounded by strong connective tissue fibres. Also, the distribution of white adipose tissue is different in males and females and is part of the secondary sexual characteristics. The storage and mobilisation of lipids does require quite some metabolic activity of the tissue. Consequently, adipose tissue has a rich supply of capillaries.

Brown adipose tissue occurs mainly during development and may account for 2 - 5 % of the body weight in a newborn. In adult individuals most of the brown fat has further differentiated into white fat. Adipocytes in brown fat contain plenty of mitochondria. A very rich capillary supply and the cytochromes found in the mitochondria give the tissue its characteristic colour. A protein (UCP-1 or thermogenin) found in these mitochondria decouples the oxidation of fatty acids from the generation of ATP. Instead, these cells generate heat.

The location of the brown fat reflects its heat-generating function. It is located in the axilla (armpits), between the shoulder blades, in the region of the neck and along large blood vessels. The heat generated by the brown fat warms the blood which supplies nearby organs or which re-enters the trunk from the limbs.


 
Suitable Slides
white adipose tissue: sections of skin - H&E, trichrome, van Gieson
Section are rarely prepared to show just white adipose tissue. The hypodermis, i.e. lightest and loosest appearing layer, of skin sections will typically contain large areas of adipose tissue. Other good candidates are bone sections which contain yellow bone marrow or sections of lymph nodes which are often embedded in adipose tissue. Small spots of adipose tissue should be present in many other sections.
brown adipose tissue: sections of brown adipose tissue or kidney - trichrome, H&E
Brown adipose tissue is often located in the connective tissue close to the renal hilus or in the renal sinus of sections which contain the entire kidney of small laboratory animals. Look for an indentation in the outline of the kidney, which corresponds to its hilus.

Thick Skin - H&E
In well-preserved thin sections, the adipocytes form a mosaic of rounded or slightly angular white tiles, which correspond to the locations of the lipid droplets, separated from each other by darker seams, which correspond to the cytoplasm of the adipocytes and the sparse connective tissue components between them. Because of the large size of the adipocytes you will only rarely see it "typical" signet ring-like appearance of the cells.
Although the tissue my be a little distorted, thicker sections give a good three dimensional feel of the adipose tissue. The cytoplasmic rims of the adipocytes form thin veils which enclose the open spaces which were occupied by the lipid droplets. Oval adipocyte nuclei are often located close to the corners at which the adipocytes meet.
Draw two or three adipocytes at high magnification and a survey image which illustrates the appearance of adipose tissue at low magnification.

 

Kidney - trichrome
In the renal sinus, islands of brown adipose tissue are often surrounded by white adipose tissue, which emphasises the different appearances of the two tissue types. In brown adipose tissue, the nuclei of adipocytes are round and located more or less centrally in a cytoplasm which, after the extraction of lipids during tissue preparation, looks very frothy. Cell borders can be difficult to identify. Capillaries are very frequent.
Sketch the appearance of the brown adipose tissue. Contrast the characteristic features of white and brown adipose if both types are present side by side.


 

Mesenchymal connective tissue

Mesenchyme forms the undifferentiated "filling" of the early embryo. It consists of mesenchymal cells, which interconnect by slender cell processes. Mesenchymal cells have stem cell properties, i.e. they are able give rise to other cell and tissues types. The wide extracellular space between the mesenchymal cells is occupied by ground substance, which can be stained with dyes that also stain mucin - hence the alternative name of this tissue type: mucoid connective tissue. Collagen or reticular fibres may not be visible at all or form a loose network between the cells. With fetal development, mesenchyme forms the connective tissue between and within the developing tissues and organs. Mucoid connective tissue also forms a compliant cushion around the vessels of the umbilical cord, where it is also called Wharton's jelly.

In adult humans, mesenchymal connective tissue is only found in the dental pulp.


 
Suitable Slides
sections of umbilical cord, tooth (pulp), or sections of embryonic and early foetal development - H&E, Azan or Alcian blue & van Gieson
section usable for "intramembranous ossification" during foetal development will contain areas of mucoid connective tissue around the developing bone.

Umbilical Cord, Human - H&E and
Foetal Kidney, Human - H&E
Within the umbilical cord you will be able to identify three large vessels and their walls. Mucoid connective tissue fills the space between the vessels and the simple squamous epithelium lining the surface of the umbilical cord. Note the very fine appearance of the collagen fibres and the lack of apparent specialisations in this type of connective tissue.
The number of cells and appearance of the collagen fibres vary depending on the precise location of the tissue. In some locations, mucoid connective tissues will contain a large number of cells and only a few, very delicate collagen fibres. Examples are dental pulp and the mucoid connective tissue which is found between the developing tubuli and glomeruli of the foetal kidney.
A small drawing should be sufficient to capture the appearance of the tissue.


page content and construction: Lutz Slomianka
last updated: 6/08/09