4. connective tissuedense regular connective tissue in tendon (outlined in yellow), showing the...

MEAT418/518 Meat Technology - 4 - 1 ©2009 The Australian Wool Education Trust licensee for educational activities University of New England

4. Connective Tissue

Graham Gardner

Learning objectives To provide a basic understanding of the structure and role of connective tissue, particularly in terms of its association with the muscular system. By the end of this lecture you should know: • The broad categorisation of different types of connective tissue. • The different types of connective tissue cells. • What constitutes the connective tissue matrix, and the type of connective tissue fibres within this

matrix. • The importance of the protein collagen, its different types, and how tropocollagen is synthesised. • The role of collagen cross-links. • The different types of connective tissue within muscle.

Key terms and concepts Collagen, elastin, extracellular matrix, endomysium, perimysium, epimysium, collagen cross-links.

4.1 Introduction Connective tissues are some of the most abundant tissues within the body, vital for providing structural strength for the entire animal. The properties and make-up of connective tissues vary throughout the body depending upon the role of the tissue that they are associated with. In terms of meat quality, this variation has a major impact on the physical attributes of different muscles, explaining a large component of the variation in tenderness evidenced between cuts of meat. The animal body is supported by bone, held together by fibrous connective tissue and protected against cold and starvation by adipose (fatty) tissue. These three types of tissue differ radically in their appearance and properties, but all are classified as connective tissue. Generally connective tissues are a supporting tissue composed of living cells embedded within a nonliving matrix that varies in consistency from fluid to crystals. Connective tissues of adult animals have relatively few cells and consist largely of extracellular matrix. The properties of these tissues are primarily due to:- • Fibrous proteins (collagen and elastin, which are extracellular). • Supporting “ground substances” (proteoglycans and glycoproteins). Each of these

components imparts visco-elastic properties to the connective tissues with their relative proportions and specific composition determining the characteristics of each tissue.

Connective tissues thus comprise the structural elements in the bodies of animals. Examples of these include: • Bones, ligaments, tendons and fascia which provide structural strength for the muscular

system. • Dermis (skin), teeth, cardiovascular system, reproductive system, digestive system, and

capsules of most organs including adipose and mucous tissues. • All the tissue in muscles other than the muscle fibres themselves.

Notes – Lecture 4 – Connective Tissue

4 - 2 – MEAT418/518 Meat Technology ©2009 The Australian Wool Education Trust licensee for educational activities University of New England

Figure 4.1 Intramuscular connective tissue. Source: Photograph taken using a scanning

microscope by I.H. Hwang.

This photo shows the structure of the intramuscular connective tissue of bovine M. longissimus dorsi, which was electrically stimulated and aged for 7 days at 1°C. The myofibre component of the muscle was digested away to leave only the connective tissue matrix. This photograph was taken using a scanning microscope. (Dr I.H. Hwang, unpublished data)

Figure 4.2 Extra-cellular matrix of connective tissue. Source:

http://www.aps.uoguelph.edu.ca/~swatland/ch2_3.htm.)

Extracellular matrix (A) of connective tissue with collagen fibre (B) and fibroblast (C).



4.2 Connective tissue types A general definition of connective tissue (CT) is that it has minimum cell-to-cell contact and maximum extracellular space. It originates in the mesenchyme and can be divided into CT proper and supportive CT. Bone and cartilage are the main types of supportive CT, and the hardness of bone results from calcification of the connective tissue matrix. Supportive CT will not be considered in depth here. CT proper is classified on the basis of the type, density and arrangement of the fibres contained in the intercellular matrix. Major types include: Loose connective tissue · Relatively rich in cells, blood vessels and nerves · Collagen fibres are common, plus some elastic and reticular fibres · Serves connective and supportive functions · Supports epithelia lining of the gut, respiratory and urinary tracts · Fills interstices between tissues

Figure 4.3 Loose irregular connective tissue. Source:

http://www.udel.edu/Biology/Wags/histopaqe/empage/ect/ect.htm.

Loose irregular connective tissue (from the Lamina propria of the small intestine). The wavy blue fibres are collagen. Dense connective tissue • completely dominated by fibres, and is subdivided into two groups according to the

arrangement of these fibres. Dense irregular connective tissue • Fibres have no clear orientation, instead forming a densely woven network. • Majority of fibres are collagen, but elastic and reticular fibres are also present. • Found in fascia, dermis, periosteum, fibrous capsules of lymph nodes, liver, and some glands.



Dense regular connective tissue • Fibres run parallel to each other • Majority of fibres are collagen • Provides great tensile strength • Found in tendons, ligaments, fascia and aponeuroses of muscles, and some organ capsules. • There is no clear demarcation between loose and dense CT, or between regular and irregular

CT.

Figure 4.4 Dense irregular connective tissue. Source: http://udel.edu/Biology/Wags/histopage/colourpage/cct/cct.htm

Dense irregular connective tissue, showing densely packed fibrils running in several directions, with few cells.



Figure 4.5 Dense regular connective tissue. Source: http://www.medsch.wisc.edu/anatomy/histo/htm/tctbase.htm

Dense regular connective tissue in tendon (outlined in yellow), showing the parallel arrangement of densely packed collagen fibres. The tendon has been severed allowing it to relax, resulting in the crimped appearance.

4.3 Connective tissue cells Mesenchymal cells All types of connective tissue are products of primitive mesenchyme, an embryonic CT derived from the mesoderm. Some mesenchymal cells remain in mature CT and provide a replacement source as needed. Fibroblasts These are the most common cell in connective tissues. They are responsible for synthesis of fibres and intercellar material. Fibroblasts are spindle shaped with cytoplasmic extensions which interlink with other fibroblasts. Macrophages These are derived from monocytes which circulate in the blood. Monocytes migrate into CT where they mature into macrophages. The major function of macrophages is defensive. Adipocytes (fat cells) Specialised connective cells adapted for the storage of fat. They vary in size (50-150mm) depending on the amount of fat contained within the cytoplasm. Mast cells and plasma cells are also found.



4.4 Connective tissue matrix This is the intercellular, non fibrous component of CT, and functions as a medium through which nutrients and waste products diffuse between capillaries and cells. It is a transparent, colourless, homogenous, and semi-fluid substance composed mostly of proteoglycans and glycoproteins. Connective tissue fibres There are two main fibres within connective tissue, collagen fibres which are composed of the protein collagen, and elastic fibres composed predominantly of the protein elastin. They display the following characteristics: Collagen fibres • 1-20 micrometers diameter • indeterminate length • unbranched • wavy in tissues which allows some capacity for stretch even though the fibres themselves are

relatively inelastic • composed of fibrils, which in turn are made up of microfibrils, which are made up of tropocollagen

molecules arranged in a staggered array

Figure 4.6 Collagen fibres. Source: http://www.udel.edu/Biology/Wags/histopaqe/empage/ect/ect.htm

Electron micrograph of collagen fibres, showing the typical wavy pattern. Elastic fibres · thinner than collagen fibres (1-4 micrometers) · thread like · highly branched · great elasticity · can form sheets in elastic ligaments · homogenous rather than fibrillar ulstructure · less prevalent in skeletal muscle



Figure 4.7 Elastin fibres. Source: http:/www.aps.uoguelph.edu.ca/~swatland/ch2_3.htm

Electron micrograph showing thin elastin fibres (stained black ). The much thicker fibres red-brown fibres are collagen. Reticular fibres • delicate collagen fibres 0.5-1 micrometers in diameter • branch to form delicate network • non-elastic • found in tissues with little tension such as glands and lymph nodes • also found in small amounts in tissues providing support for capillaries, nerves and muscle cells

4.5 Collagen Collagen is a large extracellular protein comprised of 3 polypeptide chains (called alpha chains) each of which contains long sequences of repeating tripeptides based on the general structure Gly-X-Y. Gly represents the amino acid Glycine, and is an essential component of collagen due to its small size allowing the coiled structure of the collagen polypeptide chain — glycine resides on the inner arch of the coil. X and Y can be any other amino acids, however they are most commonly proline and hydroxyproline. All collagens also contain varying quantities of the amino acids lysine and hydroxylysine, essential as they provide sites for the formation of collagen cross-links which stabilise the molecule. The collagen polypeptide alpha chain is a left-handed helix. The individual chains are unstable, but when the 3 chains are wrapped around each other they form a very stable right-handed helix characteristic of collagen. This is the tropocollagen molecule. The triple helical chains are long rod-like structures which are relatively rigid, with shorter, globular domains at each end (also called telopeptides).

Figure 4.8 Triple helix of the tropocollagen molecule,with non-helical extensions (telopeptides). Source: Gardner, (2006)



The amount of proline and hydroxyproline is directly related to the thermal stability of the helix - an important property in its physiological function, but also in respect of the properties of meat. Collagen formation Collagen formation begins with the synthesis of precursor polypeptide chains (pro-alpha-chains) within various cells including fibroblasts. Prolyl and lysyl residues within the chain are hydroxylated. Glycosylation of hydroxlysine then follows, with the amount of carbohydrate differing according to fibre type. The synthesis of the different polypeptide strands that are combined to make different types of collagen is genetically regulated. It occurs on membrane-bound polysomes within the cell, but the hydroxylation of lysine and proline occurs after the strands have been assembled. Polypeptide strands enter the cisternae of the endoplasmic reticulum, the terminal extensions of the strand are aligned, and then the strands spiral around each other, bound in this formation by disulphide bonds. These procollagen molecules have long terminal extensions protruding from the end of each newly formed triple helix. The procollagen molecules are secreted into the extracellular space. Here they are converted to tropocollagen after the terminal propeptides are cleaved (through peptidase activity) from the procollagen molecule. Outside the cell, collagen molecules become aligned in parallel formations, and they then 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. Tropocollagen is one of the longest proteins with a chain length of 300nm and a diameter of 1.5 nm. The general structure of collagen, at various levels of organisation, can be seen below. This figure shows the single strand polypeptide or alpha-chain, three of which combine to form the triple helix of the tropocollagen molecule. Tropocollagen molecules then self-assemble to form fibrils and these aggregate to form fibres.



Figure 4.9 Schematic illustration of the amino acid sequence and molecular structure for collagen. Source: Gross J. (1961)

Schematic illustration of the amino acid sequence and molecular structure for collagen and tropocollagen, and collagen fibril formation. Collagen arrangement The unique primary (tripeptide), secondary (alpha chain) and tertiary (triple helix) structure of the collagen molecule is designed to allow highly specific interactions between molecules which lead to the formation of well-defined, ordered and reproducible structures, such as the fibres found in epimysium, perimysium and endomysium. The flexible parts of each alpha chain extending beyond the triple helix (telopeptides, see Fig 4-1) are responsible for the bonding between adjacent molecules. In the polypeptide alpha chains glycin occurs at every third position. This regular distribution is responsible for the packing of tropocollagen molecules. The collagen molecules self-assemble to form fibres with a repeating pattern of 67nm, which arises from the quarter staggering of molecules in relation to their neighbours. This gives rise to overlap and gap regions which can be readily identified under electron microscopy. This can be seen in the diagram below of the arrangement of molecules in fibrils, and Photograph 4-8 which shows the appearance of collagen fibres under different magnifications. The striated pattern can be clearly seen.



Figure 4.10 Schematic drawing of collagen molecules (tropocollagen), fibrils, fibres and

bundles. Source: http://www.usd.edu/biol/faculty/swanson/histo/pics/fig16.GIF

Under the electron microscope the fibrils show a 64nm periodicity of dark and light bands, due to the stepwise overlapping arrangement of rodlike tropocollagen subunits, each measuring 280nm. This produces alternating lacunar and overlapping regions that cause the cross-striations characteristic of collagen fibrils.

Figure 4.11 Electron micrograph of collagen. Source:

http://www.udel.edu/Biology/Wags/histopaqe/empage/ect/ect.htm

Electron micrograph showing the striated pattern (right) caused by quarter staggering of tropocollagen molecules, as well as (left) the wavy structure seen under 2 magnifications. Biochemical types of collagen Each tropocollagen molecular is composed of three alpha chains but 19 unique alpha chains have so far been identified, giving rise to 11 different types of collagen Those of interest in understanding the structure of meat are shown below:



Table 4.1 Collagen types and their location in animal tissues. Source: Gardner, (2006). Type Molecular

Length (Nm) Aggregation

Characteristics Major Locations

I 300 Striated Fibres Epimysium III 300 Striated Fibres Perimysium IV 420 Non-Fibrous Endomysium

(Basement Membrane) V 300 Striated Fibres Basement Membrane

Type I: forms striated fibres between 80 and 160nm in diameter in blood vessel walls, tendon, bone, skin and muscle. It may be synthesised by fibroblasts, smooth muscle cells (around blood vessels) and osteoblasts (bone forming cells) Type III: forms reticular fibres in tissues with some degree of elasticity, such as aorta, spleen and muscle. It is synthesised by fibroblasts and smooth muscle cells, and contributes substantially to the endomysial connective tissues around individual muscle fibres, providing a small fraction of the collagen found in skin and occurs in the large collagen fibres dominated by Type I collagen. Type IV: 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, many of them are now thought to be composed of a network of irregular cords containing an axial filament of Type IV collagen. Type IV collagen occurs in the endomysium around individual muscle fibres. Instead of being arranged in a staggered array, the molecules are linked at their ends to form a loose diagonal lattice. Type V: is found prenatally in basement membranes. It has also been found in the basement membranes of muscle fibres, except at the point where muscle fibres are innervated. Collagen crosslinks and turnover Once the specific matrix structure of fibrous connective tissue has been laid down and the collagen molecules are aligned and close-packed, a second mechanism comes into play. The fibres themselves have no intrinsic strength as they are largely retained by mechanically weak non-covalent bonds. To provide tensile strength, specific lysine and hydroxylysine residues in the collagen molecules are modified by enzyme action to form aldehydes which can then form intermolecular covalent bonds called crosslinks. It is these crosslinks and their derivatives that stabilise the ordered fibres and gives them their characteristic strength, rigidity and resistance to proteolytic attack. Once these collagen structures are stabilised by crosslinks, non-enzyme reactions occur during physiological ageing of collagenous tissues which lead to further changes in the crosslinks, which then are termed mature crosslinks. Pyridinoline is a non-reducible mature cross-link which may be involved in the increased heat stability of epimysial connective tissues from older animals. Differences in the degree of cross linking may occur between different muscles of the same carcase, and between the same muscle in different species. For example, collagen from the M. longissimus dorsi has less crosslinks than collagen from the M. semimembranosus. Unlike most other proteins in muscle, collagen in connective tissue fibres has a very long half-life. An enzyme capable of degrading collagen, collagenase, is present in connective tissue but is not very active. Under some circumstances in mature animals cross-linked collagen molecules remain in place for a long time - maybe months or even years. Nevertheless, in growing muscle connective tissue structures must be remodelled to accommodate that growth. Existing connective tissue is degraded and new connective tissue is deposited. Such newly deposited connective tissue must begin the cross-linking process again. It is thought that the relative youthfulness of collagen (with consequent sparsity of mature crosslinks) in rapidly growing muscle may contribute to the tenderness of muscle from fast growing animals.



4.6 Muscle and connective tissue Connective tissue associated with the muscular system comprises: • tendons, by which muscles pull on bones • ligaments which hold bones together at joints • aponeuroses (sheets of connective tissue that attach muscles to bones or other muscles) • sheets of connective tissue that cover and separate muscles • loose connective tissue carrying blood vessels and nerves Entire muscles are wrapped in fibrous CT (epimysium). Bundles of muscle fibres called fasiculi (or fasicles) are also covered by fibrous CT (perimysium), which penetrates into the interior of each fasicle and surrounds and separates each muscle fibre (endomysium). All these are continuous with each other and are extensions of the deep fascia. They are classified as dense connective tissue, usually with regular (parallel) arrangement of fibres. The fibres are primarily collagen. Elastin fibres are present in most muscles in only small amounts. However in the M. semitendinosus and the M. latissimus dorsi, elastin makes up 30-40% of the connective tissue.

Figure 4.12 Connective tissue around muscle fibres and bundles. Source:

http:/www.aps.uoguelph.edu.ca/~swatland/ch2_3.htm

Shows the continuous mesh of connective tissue around muscle fibres and bundles. A thick layer of perimysium can be seen (A) in the photo on the right. Individual collagen fibres only lengthen by about 5% when stretched and little elasticity is possible where collagen is formed into cable-tendons. However, much of the collagen that is present in muscles forms a mesh so that stretching is possible as the configuration of the mesh, rather than collagen fibres themselves, changes. When not under tension the individual collagen fibres have a wavy or crimped appearance.



Figure 4.13 Perimysial collagen fibre arrangement. Source: Gardner, (2006).

Changes in the arrangement of perimysial collagen fibres when a muscle contracts. The arrow indicates the longitudinal axis of the muscle at rest (A) and contracted (B). In contracted muscles, most fibres of the endomysium are oriented across the muscle fibre (ie almost perpendicular to the long axis of the muscle fibre). There is often a regular periodicity and the connective tissue links adjacent muscle fibres to each other. In stretched muscles, endomysial fibres become re-aligned at an acute angle to the long axis of the muscle fibre.

Figure 4.14 Endomysial fibre arrangement. Source: Gardner, (2006).

Changes in the orientation of endomysial fibres between a contracted muscle fibre (A) and a stretched fibre (B).



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Summary Summary Slides are available on web learning management systems Connective tissues are a supporting tissue composed of living cells embedded within a nonliving matrix that varies in consistency from fluid to crystals. They provide vital structural support for the tissues of all animals, and contain large amounts of the protein collagen. Connective tissues are classified on the basis of type, broadly grouped as either loose or dense connective tissues, and are composed of 3 different types of fibres including collagen, elastic and reticular fibres. Collagen fibres are the most common and are composed of tropocollagen molecules arranged in a staggered array. This structure is stabilised by the formation of collagen cross-links which develop as an animal ages, adding to the strength and rigidity of the collagen matrix.. References Anon 2001. Gas bubbles in vacuum-packed meat. Meat Technology Update Newsletter 01/3. Food

Science Australia, Brisbane Bailey, A.J. and Light, N.D., 1989. Connective tissue in meat and meat products. Elsevier Applied

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industries: crocodiles, emus, game birds, rabbits, hares and snails RIRDC Publication No. 03/023 RIRDC Project No. DAQ 278A

Gault, N.F.S. 1992. Structural aspects of raw meat. In ‘The chemistry of muscle based foods’. (ed D.A. Ledward, D.E. Johnston, and M.K. Knight). Proceedings of a Symposium organised jointly by the Food Chemistry Group of the Royal Society of Chemistry and the Society of Chemical Industry Meat panel, Queens University of Belfast, September 1991.

Gross, J. 1961. Collagen. Scientific American, vol 204, pp 120-130. Hopkins, D.L., Walker, P.J., Thompson, J.M., Pethick, D.W. 2004b. Effect of sheep type on meat and

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sheepmeat processors Pearson and Young, R.B. 1981 Muscle and meat biochemistry. Academic Press, Principles of Meat

Science. eds M.D. Judge, E.D. Aberle, J.C. Forrest, H.B. Hedrick, R.A. Merkel, Kendall/Hunt Publishing Company, 2nd Edition 1975. pp 29-39

Sims, T.J. and Bailey, A.J. 1981. Connective tissue. In ‘Developments in Meat Science −2’, ed R.A. Lawrie. Applied Science Publishers

University of Delaware, Department of Biological Sciences Mammalian Histology, Dr. R.C. Wagner. Retrieved 25th August 2006 from http://www.udel.edu/Biology/Wags/histopage/empage/ect/ect.htm

University of Delaware, Department of Biological Sciences Mammalian Histology - B408 - Connective Tissue Ultrastructure. Retrieved October 25, 2006 from http://www.udel.edu/Biology/Wags/histopage/empage/ect/ect.htm and http://www.udel.edu?Biology/Wags/histopage/colourpage/cct/cct.htm

University of Guelph, Canada, Department of Animal and Poultry Science. Prof. H.J. Swatland. Retrieved October 25th, 2006 from http://www.aps.uoguelph.edu.ca/~swatland/ch2_3.htm

University of South Dakota, Biology Faculty, Prof. D.L. Swanson. Retrieved 25th October, 2006 from http://www.usd.edu/boil/faculty/swanson/histo/pics/fig16.GIF

University of Wisconsin, School of Medicine, Anatomy Department. Retrieved 25th August, 2006 from http://www.medsch.wisc.edu/anatomy/histo/htm/tctbase.htm

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