LEC16

16 Fibrous connective tissues

16.1 Introduction

Fibrous connective tissue are very important in meat - because they hold the myofibres together.
Some muscles have extremely strong connective tissue to prevent the myofibres being damaged as the muscle contracts.
Muscle with an angular arrangement of myofibres gain leverage when they contract. The length of the contraction is reduced but the strength is increased. The myofibres must be anchored in strong connective tissue.
The strongest connective tissues are found in the distal muscles of the limbs and in the neck.
Muscles with a high connective tissue content must be cooked with moist heat (stewed not barbecued or roasted).
Connective tissues tend to be stronger in large animals (eg., beef) than in small animals (eg., lamb).
Connective tissues tend to be stronger in old animals (eg., mature beef) than in young animals (eg., veal).
This explains why stewing beef (neck and distal limb muscles) is less expensive than steak or roasts.
This also explains why beef carcasses are graded by animal age. Grade A beef is from youthful animals (up to about 18 months of age).

Here is an image a fibrous connective tissue.

It has three essential features: (1) cells (stained with methylene blue), (2) fibres (stained pink with eosin), and (3) matrix (in the spaces between the cells and the fibres).

The fibrous connective tissues in meat form a continuous mesh, as shown in the image to the left, from the microscopic strands of endomysium around individual myofibres, to the thick layers of perimysium around bundles of myofibres (fasciculi), all being gathered and connected to the very thick epimysium on the surfaces of individual muscles.

The image below shows a thick layer of perimysium.

The endomysium, perimysium and epimysium contain two types of protein fibres, collagen and elastin, which now we will consider in detail.

16.2 Collagen fibers

Tropocollagen is an elongated protein forming extremely strong but very small collagen fibrils (best seen with an electron microscope).
Numerous collagen fibrils are bound together to form collagen fibres visible with a light microscope.
When collagen fibers form sheets or cables we can see them in meat without a microscope, and we may detect them as gristle if they do not gelatinize when meat is cooked.
Note: collagen fibres are extracellular (outside the cells forming them) whereas myofibres are cells themselves.
Tropocollagen is the most abundant protein in the animal body.
Large amounts of tropocollagen are found in animal skin. In pig skin, for example, collagen fibres are tightly woven from two directions to form a tight meshwork
Collagen is a raw material for major industries in leather, glue, cosmetics, food processing (gelatin), etc.
Under a light microscope, collagen fibres in the connective tissue framework of meat range in diameter from 1 to 12 micrometres (0.001 millimetre = 1 micrometre).
Collagen fibres do not often branch and, when branches are found, they usually diverge at an acute angle.
Collagen fibres from fresh meat are white, but usually they are stained in histological sections for examination under a microscope. The most common stain for light microscopy is eosin, which stains collagen fibres pink.
Unstained collagen fibres may be seen by polarized light since they are birefringent (with two refractive indices like A bands). By rotating the plane of polarized light, collagen fibres appear bright against an otherwise dark background (when two Polaroid lenses are perpendicular they block most of the light, but collagen fibres can rotate the light so they appear bright).
The birefringence of collagen fibres in meat is lost during cooking at the point gelatinization occurs.
Collagen fibres have a wavy or crimped appearance which disappears when they are placed under tension.
Collagen fibres fluoresce with a blue-white light when excited with UV light enabling the amount of connective tissue on a cut meat surface to be measured very rapidly. Peak excitation is around 370 nm so that the prominent 365 nm peak emission of a mercury arc lamp may be used. Some indication of collagen fibre diameter may be obtained by spectrofluorometry (measuring the wavelengths of fluorescence) because the fluorescence is quenched (fades) fairly rapidly. Thus, large collagen fibres retain a central core with a pre-quenching emission spectrum for longer than small fibres. Fat only fluoresces weakly, to about the same extent as areas of muscle with a low connective tissue content. Collagen fluorescence increases with animal age.
Electron microscopy shows collagen fibrils with diameters ranging from 20 to 100 nm (0.001 micrometre = 1 nanometre). Collagen fibrils typically have diameters which are multiples of 8 nm showing the manner in which they grow radially.
Collagen microfibrils (even smaller structures that make up fibrils) may appear to have a tubular structure with an electron-lucent lumen (appearing empty under the electron microscope).
Collagen fibrils are formed from long tropocollagen molecules which are staggered in arrangement but tightly bound laterally by covalent chemical bonds.
For electron microscopy, when negatively stained with heavy metals, the stain spreads into the spaces between the ends of molecules, and collagen fibrils appear to be transversely striated.
The periodicity of these striations is 67 nm but often shrinks to 64 nm as samples are processed for examination
Collagen fibres are formed by cells called fibroblasts.
Although collagen fibres are located outside the cell, the initial stages of collagen fibril assembly may be within the cell, with fibril morphology being regulated by a special site on the fibroblast membrane.

Tropocollagen is a high molecular weight protein (300,000 Daltons) formed from three polypeptide strands twisted into a triple helix. Each strand is a left-handed helix twisted on itself, but the three strands are twisted into a larger right-handed triple helix. The triple helix is responsible for the stability of the molecule and for the property of self-assembly of molecules into microfibrils. The flexible parts of each strand projecting beyond the triple helix (telopeptides) are responsible for the bonding between adjacent molecules. In other words, the cross links binding tropocollagen molecules together laterally are made between the helical shaft of one molecule and the non-helical extension of an adjacent molecule.

In the polypeptide strands, the small amino acid glycine occurs at every third position, and proline and hydroxyproline account for 23% of the total residues. The regular distribution of glycine is required for the packing of tropocollagen molecules and has been claimed as evidence all animals are derived by evolution from a single ancestral stock, since the chance development of this unique regularity in unrelated animals is thought unlikely. Hydroxyproline is quite rare in other proteins of the body, and an assay for this imino acid (an imino acid is chemically similar, but not the same as an amino acid) provides a measure of the collagen or connective tissue content in a meat sample. Tropocollagen also contains a fairly high proportion of glutamic acid and alanine as well as some hydroxylysine.

16.4 Biochemical types of collagen

The various types of collagen of interest in understanding the structure of meat are as follows.

Type I collagen forms striated fibres between 80 and 160 nm in diameter in blood vessel walls, tendon, bone, skin and meat. It may be synthesized by fibroblasts, smooth muscle cells (around blood vessels) and osteoblasts (bone-forming cells).
Type II collagen fibres are less than 80 nm in diameter and occur in hyaline cartilage (for example, in the soft blade of the scapula) and in intervertebral discs. It is synthesized by chondrocytes (cartilage-forming cells).
Type III collagen forms reticular fibres in tissues with some degree of elasticity, such as spleen, aorta and muscle. It is synthesized by fibroblasts and smooth muscle cells, contributes substantially to the endomysial connective tissues around individual myofibres, provides a small fraction of the collagen found in skin and occurs in the large collagen fibres dominated by Type I collagen. It may have some function in regulating collagen fiber growth. Unlike Type I collagen fibres, reticular Type III fibres are highly branched.
Type IV collagen occurs in the basement membranes around many types of cells and may be produced by the cells themselves, rather than by fibroblasts. Although basement membranes were once regarded as amorphous (like glue), many of them now are thought to be composed of a network of irregular cords. The cords contain an axial filament of Type IV collagen, ribbons of heparin sulphate, proteoglycan, and fluffy material (laminin, entactin and fibronectin). Type IV collagen occurs in the endomysium around individual muscle fibers. Instead of being arranged in a staggered array, the molecules are linked at their ends to form a loose diagonal lattice.

Tendons often extend into the belly of a muscle or along its surface before they merge with its connective tissue framework, and Types I and III collagen both may be extracted from meat. Even within tendons, there may be some Type III collagen forming the endotendineum or fine sheath around bundles of collagen fibrils. In fibres composed of collagen Types I and II, fibrils have a straight arrangement whereas, in fibres of Type III collagen, the fibrils have a helicoidal arrangement.

Small diameter Type III collagen fibres are called reticular fibers since, when stained with silver for light microscopy, they often appear as a network or reticulum of fine fibres. The larger diameter collagen fibres formed from Type I collagen are not blackened by silver.

Collagen fibres shrink when they are placed in hot water, and ultimately they may be converted to gelatin. Around 65ºC, the triple helix is disrupted and the alpha chains fall into a random arrangement. The importance of this change? It tenderizes meat with a high connective tissue content.

Tropocollagen molecules from older animals are more resistant to heat disruption than those from younger animals.

16.4 Collagen biosynthesis

The synthesis of the different polypeptide strands combining to make different types of tropocollagen is genetically regulated by the production of messenger RNA. The synthesis of polypeptide strands occurs on membrane-bounded polysomes, but the hydroxylation of lysine and proline occurs after the strands are assembled. Ascorbic acid is required for the hydroxylation of lysine and proline. Polypeptide strands enter the cisternae of the endoplasmic reticulum (a membranous assembly labyrinth within the cell), the terminal extensions of the strands are aligned, and then the strands spiral around each other. Procollagen or immature tropocollagen has long terminal extensions protruding from each end of the newly formed triple helix. Procollagen moves to the golgi apparatus and is packaged into vesicles moved to the cell surface, probably by microtubules. Except for some Type III procollagen molecules, the long terminal extensions are then enzymatically reduced in length.

Outside the cell, tropocollagen molecules become aligned in parallel formations, and then they link up laterally to form fibrils. It is likely that tropocollagen monomers are partially assembled together in groups before they are added to an existing collagen fibril. Firstly, vacuoles containing procollagen fuse to form a fibril-containing compartment. Then the cytoplasmic extensions withdraw from between several fibril-forming compartments to create a bundle-forming compartment.

16.5 Crosslinking of tropocollagen molecules

Within an individual tropocollagen molecule, the three polypeptide strands are linked together by stable intramolecular bonds originating in the non-helical ends of the molecule.
The great strength of collagen fibres, however, originates mainly from the stable intermolecular covalent bonds between adjacent tropocollagen molecules.
Stable disulphide bonds between cystine molecules in the triple helix also occur.
During the growth and development of meat animals, covalent cross links increase in number, and collagen fibres become progressively stronger.
Meat from older animals, therefore, tends to be tougher than meat from the same region of carcasses from younger animals.
This relationship is complicated in young animals by the rapid synthesis of large amounts of new tropocollagen. New tropocollagen has fewer cross links so, if there is a high proportion of new tropocollagen, the mean degree of cross linking may be low, even though all existing molecules are developing new cross links.
As the formation of new tropocollagen slows down, the mean degree of cross linking increases.
Another complication is many of the intermolecular cross links in young animals are reducible (the collagen is strong but is fairly soluble).
In older animals, reducible cross links are converted to non-reducible cross links (the collagen is strong but is far less soluble and more resistant to moist heat).
Pyridinoline is a non-reducible cross-link causing increased heat stability of connective tissues from older animals. It is formed without enzymes by glycosylation (a reaction between lysine and reducing sugars).
Differences in the degree of cross linking may occur between different muscles of the same carcass, and between the same muscle in different species.
Nutritional factors such as high-carbohydrate diet, fructose instead of glucose in the diet, low protein, and pre-slaughter feed restriction may reduce the proportion of stable cross links.
The rate of collagen turnover in skeletal muscle may be about 10% per day and the turnover time for collagen may be inversely proportional to collagen fibril diameter.

16.6 Elastic fibres & elastin

Individual collagen fibres only lengthen by about 5% when stretched and little elasticity is possible where collagen is formed into cable-like tendons. However, much of the collagen present in meat forms a meshwork and stretching of the whole meshwork is possible because its configuration changes. Fibres with truly elastic properties, however, are necessary in structures such as the ligamentum nuchae of the neck and in the abdominal wall. And all arteries, from the aorta down to the finest microscopic arterioles, rely on elastic fibres to accommodate the surge of blood from contraction of the heart. Elastic fibres may be stretched to several times their original length but rapidly resume their original length once released. Elastic fibres are composed of the protein, elastin. Elastin is found in all vertebrates except primitive jawless fish, and in evolution it appeared first in cartilaginous fish. The elastic fibers in the image below (from loose connective tissue around the intestine) are the thin black ones. The much thicker, red-brown fibres are collagen.

Elastin resists severe chemical conditions, such as the extremes of alkalinity, acidity and heat destroying collagen.

Fortunately, there are relatively few elastic fibres in muscle, otherwise cooking would do little to reduce meat toughness.
The elastic fibres in muscles used frequently for locomotion are larger and more numerous than those of less frequently used muscles.
Elastic fibres in the epimysium and perimysium of beef muscles range from 1 to 10 microns in diameter.
Elastic fibres usually are pale yellow.
When elastic fibres are stretched, they may become visible in polarized light without staining.
Elastic fibres in the connective tissue framework of meat are usually branched.
Electron microscopy reveals elastic fibres are composed of bundles of small fibrils approximately 11 nm in diameter embedded in an amorphous material.
Although elastin resembles tropocollagen in having a large amount of glycine, it is distinguished by the presence of two unusual amino acids, desmosine and isodesmosine.
Like collagen, elastin contains hydroxyproline, although it may not have the same function of stabilizing the molecule.
Tropoelastin, the soluble precursor molecule of elastin (molecular weight 70,000 to 75,000 Daltons), is secreted by fibroblasts after it has been synthesized by ribosomes of the rough endoplasmic reticulum and processed by the Golgi apparatus.
In the presence of copper, lysyl oxidase links together four lysine molecules to form a desmosine molecule.
Isodesmosine is the isomer of desmosine.
The aorta may be fatally weakened by a lack of mature elastin in animals deprived of dietary copper.
Elastin in the arterial system is produced by smooth muscle cells instead of fibroblasts.

16.7 Why meat must be a natural food for us

During the digestion of meat in the human gut, elastic fibres are broken down by elastase, an enzyme from the pancreas. We would not have this enzyme if our evolutionary ancestors had not been at least partly carnivorous. In other words, I have never read of the occurrence of elastin in any human food except meat. So if we have evolved a highly specific enzyme, elastase, to deal with elastin in our food, this can only mean that we are the descendants of meat eaters.

Further information

Structure and Development of Meat Animals and Poultry. Pages 82-95.