Connective tissue

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Living organisms are not merely random collections of cells; each organism grows according to a detailed pattern that determines both the structure and the functions of its many constituent parts, and ensures that they are all integrated with one another both anatomically and biochemically. One of the basic requirements for systematic arrangement within tissues is a mechanical one—a means of maintaining the cells in appropriate relationship to each other despite the various forces that act upon them, including gravity, externally applied physical stress, and the internal movements of the organism itself, which result from growth and exertion. Two contrasting systems for organizing their mechanical structures are employed by multicellular organisms. These systems differ in whether the mechanical elements that determine the rigidity of the structure are closely associated with cells or frankly extracellular. This chapter describes the characteristics and functions of the connective tissue. They fulfill many functions but their primary function is a mechanical one, connective tissue elements being responsible for maintaining cells, tissues, and organs in proper relationship to one another. They also provide the animal with support, usually by means of a rigid skeleton, to which the softer tissues are attached, and with a system for the transmission of mechanical force, so that the contractile power of the muscles can be harnessed to the skeletal framework and used to move the animal as a coordinated whole.

Living organisms are not merely random collections of cells; each organism grows according to a detailed pattern which determines both the structure and the functions of its many constituent parts, and ensures that they are all integrated with one another both anatomically and biochemically. One of the objects of this book is to emphasize the molecular basis of life and, further, that chemical and anatomical structures merge into each other, these terms merely serving to distinguish different orders of size and perhaps complexity. There can be no better illustration of the merging of these two orders of structure than the connective tissues.

One of the basic requirements for systematic arrangement within tissues is a mechanical one – a means of maintaining the cells in appropriate relationship to each other despite the various forces which act upon them, including gravity, externally applied physical stress and the internal movements of the organism itself which result from growth and exertion.

Two contrasting systems for organizing their mechanical structures are employed by multicellular organisms. These systems differ in whether the mechanical elements that determine the rigidity of the structure are closely associated with cells or frankly extracellular. A good example of a mechanical system of the former type occurs in higher plants where the cells secrete a strong thick layer around themselves, the cell wall, which resists the internal pressure exerted against it by the cell itself. As a result, the individual cells are rigid like inflated motor tyres and thus form the basis of a rigid multicellular structure. The main building material of plant cell walls is cellulose, a homopolymer composed of bundles of chains each of which contains about 5000 glucose units. Another kind of cell-based mechanical organization occurs in the keratinous epithelia of higher animals (Chapter 26).

The cellular type of mechanical organization has certain limitations and is suitable mainly for static tissue situations. Animals lead active lives and their mechanical structures are adapted to various dynamic requirements, both in the stresses they have to meet and in the changes which tissues undergo in the course of a lifetime. As a result, in many animal tissues, the cells have lost their primary mechanical function and occupy a very much smaller proportion of the total volume of the tissue than in plants. In animal tissues the mechanical function is taken over by extracellular tissue elements, comprising protein fibres embedded in a polymeric aqueous gel. The restriction of mechanical function to extracellular tissue components permits larger spaces between cells and makes possible a greater freedom in tissue pattern. Thus more than one type of cell may occur in the same tissue and there is scope for continuous reorganization to meet the needs of growth, repair, changing stress and cell replacement, without loss of the original tissue characteristics. All these features are seen in an extensive but unified family of tissues found in vertebrates, known as the connective tissues.

Occurrence and characteristics of connective tissues

Connective tissues fulfil many functions but their primary function is a mechanical one, connective tissue elements being responsible for maintaining cells, tissues and organs in proper relationship to one another. They also provide the animal firstly with support, usually by means of a rigid skeleton, to which the softer tissues are attached and, secondly, with a system for the transmission of mechanical force, so that the contractile power of the muscles can be harnessed to the skeletal framework and used to move the animal as a coordinated whole. Connective tissues are essentially mesodermal in origin being derived from the primitive mesenchyme, a layer of the early embryo. They all contain the fibrous protein collagen, as their most important and characteristic constituent. Typical forms are tendon, the corium layer of the skin, loose connective tissue, cartilage and the basement membranes of various tissues. In a mineralized form connective tissue is present in calcified cartilage, bone, dentine and cementum, which are described in Chapter 29. Connective tissues thus include many of the tissues of specific dental interest such as the oral subepithelium (corresponding to the corium of skin), the periodontal membrane or periodontal ligament, the alveolar bone of the tooth socket, cementum, dentine and the dental pulp.

The main components of connective tissues are shown in Table 27.1. The fibroblasts are responsible for the synthesis of the remaining non-living extracellular components, and, when actively laying down collagen fibres in soft connective tissues, are typically amoeboid cells which protrude spiky processes. They may become differentiated as in bone, where the osteocytes become embedded throughout the mineralized tissue, and in dentine, where odontoblasts remain near the boundary between the mineralized dentine and the pulp with their processes passing through the thickness of the dentine. The connective tissue cells first lay down the ground substance as a gel containing various kinds of macromolecule. This contains a relatively large volume of water and provides a suitable environment for the subsequent deposition of the fibrous proteins. These are secreted by the fibroblasts as macromolecules which are further assembled extracellularly, and eventually give rise to large insoluble fibrous aggregates, which, as the connective tissue matures, fill most of the extracellular space and give the tissue great mechanical strength.

Table 27.1

Components of connective tissues

Cells	Fibroblasts in more or less differentiated forms, e.g. chondroblasts, odontoblasts, osteocytes, osteoblasts, etc.
	Fat cells, macrophages, plasma cells, mast cells and leucocytes
Interfibrillar matrix or ground substances (molecules, often high polymers)	Proteoglycans (glycosaminoglycans)
	Glycoproteins
	Phospholipids
	Water
Protein fibres	Collagen
	Reticulin
	Elastin

Although the most common and characteristic type of connective tissue cell is the fibroblast many other types of cell are present in loose connective tissue. These mostly have a protective role. They include fat cells, macrophages, plasma cells, mast cells and leucocytes. The macrophages and some of the leucocytes are phagocytic and are able to engulf cellular debris, bacteria and inert foreign matter; the plasma cells are derived from B-lymphocytes and produce immunoglobulins while the mast cells produce heparin and histamine and also, in some species, serotonin (5-hydroxytryptamine).

The ground substance

A great variety of complex substances, of high molecular weight which contain both sugar and amino acid units, are associated with the fibrous elements of connective tissues. These are collectively described as mucosubstances or glycoconjugates, and may be divided into two categories known as proteoglycans and glycoproteins. Some lipid material is also present.

The proteoglycans contain heteropolysaccharides known as glycosaminoglycans, an unwieldy term that emphasizes their content of hexosamines and uronic acids. Formerly glycosaminoglycans were known as mucopolysaccharides. The best known members of this group are the chondroitin sulphates and hyaluronic acid. Though they are characterized by the structure of their polysaccharide chains, these are almost invariably associated with protein, hence the term proteoglycan.

Proteoglycans

The most characteristic structural feature of the proteoglycans is the possession of very highly polymerized carbohydrate chains (with 150 to several thousand sugar residues) having only two kinds of modified sugar residue alternating along the whole length of the chain. One of these is usually a hexosamine and the other, a hexuronic acid. Hexosamines are derived from hexose sugars by replacement of the hydroxyl group on C-2 by an amino group. Sometimes this amino group is modified by having an attached acetyl group so that the resulting structure (−NH·CO·CH₃) does not ionize and cannot acquire a positive charge. Hexuronic acids resemble the hexose sugars but differ from them in having C-6 as a carboxyl group instead of a primary alcohol group. Provided that the carboxyl group is unsubstituted, it confers weakly acid properties. In some proteoglycans, a molecule of sulphuric acid is attached in ester linkage to an oxygen atom from one of the hydroxyl groups of the hexosamine. Ionization of the ester sulphate group gives such proteoglycans strongly acidic properties, which are responsible for their specific staining reaction with the dye Alcian Blue. Thus proteoglycans are large polyvalent anions, and are able to attract and bind cations, usually Na⁺ and K⁺, referred to as counterions which maintain both electrical and chemical neutrality.

With the exception of keratan sulphate, which contains galactose residues in place of uronic acid, the composition of connective tissue glycosaminoglycans shows variations on the theme described in the previous paragraph (Table 27.2). Hyaluronic acid is only weakly acidic, whereas the chondroitin sulphates have strongly acidic sulphate groups.

Table 27.2

Features of the composition of glycosaminoglycans from connective tissues

Polysaccharide	Hexosamine	Uronic acid (or hexose)
Hyaluronic acid	N-Acetyl-D-glucosamine (1:3 linkage, SO₄ absent)	D-Glucuronic acid (1:4 linkage)
Chondroitin 4-sulphate	N-Acetyl-D-galactosamine (1:3 linkage, 4-SO₄)	D-Glucuronic acid (1:4 linkage)
Dermatan sulphate	N-Acetyl-D-galactosamine (1:3 linkage, 4-SO₄)	L-Iduronic acid (1:4 linkage)
Chondroitin 6-sulphate	N-Acetyl-D-galactosamine (1:3 linkage, 6-SO₄)	D-Glucuronic acid (1:4 linkage)
Chondroitin	N-Acetyl-D-galactosamine (1:3 linkage, SO₄ absent)	D-Glucuronic acid (1:4 linkage)
Heparan sulphate	N-Acetyl-D-glucosamine (partially sulphated)	D-Glucuronic acid
Keratan sulphate	N-Acetyl-D-glucosamine (1:4 linkage, 6-SO₄)	D-Galactose (1:3 linkage)

Glycosaminoglycans are nearly always associated with a smaller amount of protein. At one time this was thought to be a loose electrostatic association, but recently it has been shown that the polysaccharide is usually covalently linked through an alkali-labile O-glycosidic bond to a serine group on the protein. This gives a very large molecule, with a single protein chain, the core protein, to which many long carbohydrate chains are covalently attached (Figure 27.1).

Figure 27.1 Arrangement of a proteoglycan molecule with protein core and glycosaminoglycan chains

Hyaluronic acid occurs widely in connective tissues but is most easily isolated from synovial fluid which acts as a lubricant for the cartilage of joints, or from umbilical cord where it exists as a complex containing 25–30% of protein. The association is a loose one as removal of protein does not cause a marked fall in the high viscosity of synovial fluid. The particle weight of hyaluronic acid is very high being 1–4 × 10⁶, the structure consisting of a single very long polysaccharide chain. Mutual electrostatic repulsion between the negatively charged carboxyl groups on the uronic acid residues causes the molecule to form a loosely tangled skein or net in intimate contact with the extracellular water. Like other proteoglycans, it does not possess a unique configuration, the flexible molecules having ‘average’ shapes.

Chondroitin sulphate is also found in small amounts in many connective tissues and is a major constitutent of cartilage. The material from cartilage has a molecular weight of 1–5 × 10⁶ and the molecule consists of a protein core with 30–60 polysaccharide chains, each of molecular weight approximately 50000, covalently attached. Mutual repulsion of the negatively charged carbohydrate chains makes the core behave like a rigid rod 3700 Å (370 nm) in length.

Glycoproteins

Glycoproteins are conjugated proteins having one or more short irregular heterosaccharide side chains bound covalently to the polypeptide chain. In some respects therefore they resemble proteoglycans of the chondroitin sulphate type but differ from them in the shortness of their polysaccharide chains (2–20 units long) which do not contain a regularly repeating disaccharide unit. The carbohydrate content is usually lower than in proteoglycans. Glycoproteins vary enormously in the number, composition and size of their carbohydrate side chains. Like glycosaminoglycans, they possess acetylhexosamines but uronic acids are not present in glycoproteins. Mannose and galactose and the methyl pentose, fucose (6-deoxy-L-galactose), occur commonly, but the most characteristic constituent is N-acetylneuraminic acid (sialic acid) which often terminates the free ends of the carbohydrate chains. Sialic acid is both a sugar and an amino acid and its strongly acid carboxyl group (pK 2·6) gives it acidic properties throughout a wide range of pH, which it confers on the glycoprotein as a whole. Glycoproteins which contain sialic acids are known as sialoproteins. The polar nature of sialic acid makes this group of proteins strongly hydrophilic, stretching out the side chains in an aqueous environment. Glycoproteins are present in saliva and are responsible for its surface active and ‘spinnbarkeit’ properties. They probably occur in the ground substance of many connective tissues but in rather small proportions. The best characterized connective tissue glycoprotein is bone sialoprotein (page 439).

The glycoproteins, as mentioned in Chapter 5, are a most interesting and varied group of proteins and are of special importance in oral biology as constituents of both saliva and the capsules of many bacteria. The rigid wall of these bacteria is composed of the glycopeptide murein, which is elaborately cross-linked so that it forms a single bag-shaped molecule. This is responsible for the shape and mechanical strength of the cell wall (page 205) and its importance is illustrated by the lethal effects of penicillin, which interferes with its synthesis (page 480). Its carbohydrate component is susceptible to the antibacterial enzyme lysozyme.

The main classes of lipid identified in connective tissues include neutral fats, fatty acids, phospholipids and sterols, especially cholesterol. Very few investigations have been made on lipids in soft connective tissues, except for their deposition in porous foreign bodies implanted in these tissues. These experiments suggest that all four types of lipid can be synthesized by fibroblasts.

Functions of the ground substance

An essential function of the proteoglycans in the ground substance of connective tissues is to provide an extracellular environment which facilitates the laying down and maturation of fibres during tissue development. Newly formed tissues, whether in the embryo or young animal or the granulation tissue in healing wounds, have larger proportions of water and carbohydrate and smaller amounts of protein, especially collagen fibres, than older tissues. This may explain why it is difficult for sutures to hold in young and healing tissues.

The higher water content of young tissues provides a clue to one of the most important properties of proteoglycans, which enables them to form the intial extracellular milieu for fibre deposition. Their molecules being polyanionic spread out in net-like form, each strand being associated with a comparatively large quantity of water. This is hydrogen bonded to the hydrophilic hydroxyl groups and loosely held by ‘solvation’ of the fixed ionized groups on the proteoglycan chains (see Chapter 3). Because they have very open structures the proteoglycans will hold an enormous weight of water per unit weight of organic material and, although soft tissues contain up to 70% of water, no fluid seeps out when they are punctured.

The small molecules of nutrients and waste products can diffuse freely, through the water held by proteoglycans, but diffusion of macromolecules, such as tropocollagen (page 44), is restricted to those regions that are fairly remote from the individual strands of this hydrated net. Such molecules are therefore channelled through the free water, either within the large holes in the network or between the larger sheets of netting. Presumably the restriction to free diffusion resulting from the size and spatial distribution of these holes affects the distribution and orientation of newly laid down collagen fibres.

Evidence for the existence of proteoglycan nets, the strands of which are surrounded by water envelopes, was obtained by subjecting a gel of soluble collagen to ultracentrifugation. This produced a compact pellet of collagen containing very little water, but when the experiment was repeated with the addition of a very small proportion of hyaluronic acid to the collagen, a much larger pellet with a higher water content was obtained. It was calculated that the thin chains of hyaluronic acid are normally surrounded by an envelope of water having a radius of 50 Å (5 nm) from which the soluble collagen was excluded. Chondroitin sulphate has an even greater effect than hyaluronic acid and can retain ten times more water than a pellet containing collagen alone. It has been suggested that collagen may become entangled in the fine polysaccharide side chains of chondroitin sulphate.

At present it is not possible to account for the specific behaviour of each of the various proteoglycans occurring in tissues. The kinds and proportions of different mucosubstances vary greatly between tissues and even change in the same tissue as it ages. Thus cartilage is rich in chondroitin 4-sulphate and dermatan sulphate, whereas chondroitin 6-sulphate and keratan sulphate are dominant in the cornea. Skin contains hyaluronic acid, dermatan sulphate and chondroitin 6-sulphate, which predominate in turn as the embryonic tissue grows older. As rib cartilage ages, the chondroitin sulphate is replaced by keratan sulphate. The proportion of fibrous protein to proteoglycan also varies in different tissues and, as already mentioned, increases with age. Since collagen is the principal fibrous protein and characteristically contains a high proportion of hydroxyproline (page 35), the hydroxyproline/hexosamine ratio is a convenient index of the extent to which the ground substance has been impregnated with fibres. Values for this index vary considerably, e.g. 2·8 for cartilage, 12·2 for skin and 30 for tendon.

The various constituents of connective tissues are structurally integrated and do not act in isolation. Thus although cartilage is characteristically rich in proteoglycans, very little can be extracted with water but, if the tissue is finely ground to destroy the structure, as much as 60–80% of the total chondroitin sulphate can be extracted. The remaining polysaccharide is firmly bound to the collagen and can only be removed by reagents that break bonds between carbohydrate and protein. These bonds help to anchor proteoglycans in position within the tissue.

The connective tissue glycosaminoglycans are readily broken down within the body and numerous data on the turnover rates of hyaluronic acid and the chondroitin sulphates have shown their half-lives to vary from 2 to 4 days depending on the type of glycosaminoglycan, the organ and the age of the animal.

Enzymes known as hyaluronidases are widely distributed in animal tissues and, in several cases, have been shown to have acid pH optima and to be localized in the lysosomes. Experiments carried out in vitro have, in fact, shown the lysosomes to contain a full complement of the enzymes needed to convert hyaluronic acid and the sulphated glycosaminoglycans to their monosaccharide constitutents. The enzymes of this hyaluronidase group are able to depolymerize the glycosaminoglycans in vivo, so facilitating movement of both water and polysaccharide through the tissue spaces. By locally breaking down this attenuated structure the hyaluronidases increase the permeability of the tissue and when present in animal venoms and bacterial toxins they act as ‘spreading factors’. The oligosaccharides produced by the action of hyaluronidase may subsequently be acted upon by various exoglycosidases.

Some disorders of proteoglycan metabolism, e.g. Hurler’s syndrome, have been shown to be enzyme-deficiency diseases in which a lysosomal enzyme is missing (page 203).