Connective tissue

Structural composition

Together with muscle, nerve and epithelium, connective tissue is one of the basic components in the human body. It binds structures together, helps in mechanical and chemical protection and also plays a principal role in reparative processes.

Connective tissues are defined as those composed predominantly of the extracellular matrix and connective tissue cells. The matrix is made up of fibrous proteins and a relatively amorphous ground substance. Many of the special properties of connective tissues are determined by the composition of the matrix, and their classification is also largely based on its characteristics.

Connective tissue cells

Cells of general connective tissues can be separated into the resident cell population (mainly fibroblasts) and a population of migrant cells with various defensive functions (macrophages, lymphocytes, mast cells, neutrophils and eosinophils), which may change in number and moderate their activities according to demand.

Fibroblasts, the majority of cells in ordinary connective tissue, arise from the relevant undifferentiated mesenchymal stem cells ¹ and are involved in the production of fibrous elements and non-fibrous ground substance (Fig. 3.1). During wound repair they are particularly active and migrate along strands of fibrin by amoeboid movements to distribute themselves through the healing area to start repair. Fibroblast activity is influenced by various factors such as the partial pressure of oxygen, levels of steroid hormones, nutrition and the mechanical stress present in the tissue.²

Fig 3.1 Electron micrograph of a fibroblast in human connective tissue, surrounded by bundles of finely banded collagen fibrils (shown at high magnification in the insert) which they secrete. From Standring, Gray’s Anatomy, 40^th edn. Churchill Livingstone, Edinburgh, 2008 with permission.

The other cell types are migrant cells and only occasionally present, such as: macrophages, lymphocytes, mast cells, and granulocutes (Table 3.1).^3,4

Table 3.1

Connective tissue cell types

Box 3.1 Components of connective tissue

Cells

• fibroblasts → fibrous connective tissue

• chondrocytes → cartilage

• osteoblasts and osteocytes → bone

Extracellular matrix (ECM)

• fibres: collagen → framework of the ECM

• elastin → extensible element of ECM

• proteoglycans: hydrators, stabilizers and space fillers of ECM

• glycoproteins: stabilizers and linkers of ECM

• fluid

The basic molecule of collagen is procollagen, synthesized in the fibroblast, illustrated in Figure 3.3, steps 1–4. It is formed of three polypeptide chains (α-chains). Each chain is characterized by repeating sequences of three amino acids – glycine, proline and lysine joined together in a triple helix. The helical molecules are secreted into the extracellular space where they slowly polymerize and crosslink (Fig. 3.4). They overlap each other by a quarter of their length, lie parallel in rows and are collected into large insoluble fibrils. The fibrils unite to form fibres, finally making up a bundle. An aggregate of bundles makes up a whole structure such as a ligament or a tendon. The individual bundles are in coils, which increases their structural stability and resilience, and permits a small physiological deformation before placing the tissue under stress, and in consequence permits a more supple transfer of tractive power in the structure itself and at points of insertion (Fig. 3.5). The process of collagen synthesis is stimulated by some hormones (thyroxine, growth hormone and testosterone), although corticosteroids reduce activity.

Fig 3.3 The successive steps in collagen synthesis by fibroblasts.

Fig 3.4 Crosslinking and interspaces between head and tail of neighbouring tropocollagen molecules. They overlap each other by a quarter of their length. Molecules in the same parallel row are separated from each other by small interspaces.

Fig 3.5 (a) Unloaded collagen fibres in a human knee ligament. (b) Physiological deformation after stress. From Kennedy et al⁷ with permission (http://jbjs.org/).

Connective tissue collagen can be classified into different types of which at least 14 are now genetically characterized and the others are being investigated. In the context of this book the most important are:

• Type I: the most abundant of all collagen. Strong thick fibres packed together in high density. It predominates in bone, tendon, ligament, joint capsule and the annulus fibrosus of the intervertebral disc.

• Type II: thin fibres found in articular cartilage and the nucleus pulposus of the intervertebral disc. They particularly function in association with a high level of hyaluronan and sulphated proteoglycans to provide a hydrated and pressure-resistant core.

• Type III: essentially present in the initial stages of wound healing and scar tissue formation. It secures early mechanical strength of the newly synthesized matrix. These relatively thin, weak fibres are replaced by the strong type I fibres as healing proceeds.

In relation to the degree of orientation of fibrous tissue elements, ordinary connective tissues can also be classified into regular and irregular types.

Regular types

Highly fibrous tissues such as ligaments, tendons, fascia and aponeuroses are predominantly collagenous and show a dense and regular orientation of the fibres with respect to each other. The direction of the fibres is related to the stress they experience. Collagen bundles in ligaments and tendons are very strong and rupture usually takes place at the bony attachments rather than by tearing within their substance (Fig. 3.6).

Fig 3.6 Dense regular connective tissue in a tendon. Thick parallel bundles of type 1 collagen (asterisks) give tendon its white colour in life. The elongated nuclei of inactive fibroblasts (tendon cells) are visible between collagen bundles. From Standring, Gray’s Anatomy, 40^th edn. Churchill Livingstone, Edinburgh, 2008 with permission.

Irregular types

The irregular types consist of collagen and elastin interlacing in all directions. It is loose, extensible and elastic and found between muscles, blood vessels and nerves. It binds partly together, although allowing a considerable amount of movement to take place. In the sheaths of muscles and nerves and the adventitia of large blood vessels, the tissue is more dense with a high proportion of collagen fibres to protect these structures against considerable mechanical stress. The dura mater is also an example of an irregular connective tissue sleeve.

Vascularization

Connective tissue is poorly supplied with blood vessels. In dense fibrous tissues these usually run parallel to and between the longitudinal bundles with communicating branches across these.

Lymphatic vessels are more numerous, especially in loose connective tissues such as the dermis. They are also abundant in tendons and tendon sheaths.

Innervation

Dense connective tissues, for example, ligaments, tendons and fascia, have a rich supply of afferent nerve endings. The various sensory receptors transmit information to the central nervous system about changes in length and tension which allows constant monitoring of the position and movement of a joint as well as of injurious conditions that threaten these structures.

Structural and physiological studies 8,9 have shown the presence of at least four types of receptor. Three of these have encapsulated endings but the fourth consists of free unencapsulated endings:

• Type 1 (Ruffini endings) are present in the superficial layers of a fibrous joint capsule. They respond to stretch and pressure within the capsule and are slow adapting with a low threshold. They signal joint position and movement.

• Type 2 are particularly located in the deep layers of the fibrous capsule. They respond to rapid movement, pressure change and vibration but adapt quickly. They have a low threshold and are inactive when the joint is at rest.

• Type 3 are found in ligaments. They transmit information on ligamentous tension so as to prevent excessive stress. Their threshold is relatively high and they adapt slowly. They are not active at rest.

• Type 4 are free unencapsulated nociceptor terminals which ramify within the fibrous capsule, around adjacent fat pads and blood vessels. They are thought to sense excessive joint movements and also to signal pain. They have a high threshold and are slow adapting. Synovial membrane is relatively insensitive to pain because of the absence of these nerve endings.

All these receptors influence muscle tone via spinal reflex arcs which are formed by the same nerves that supply the muscles which act on the joint. Parts of the joint capsule supplied by a given nerve correspond with the antagonist muscles. Tension on this part of the capsule produces reflex contraction of these muscles to prevent further overstretching of the capsule. In consequence all receptors have an important function in stabilization and protection of the joint. After rupture of a capsule, ligament perception is considerably disturbed because of the disruption of the transmission of afferent information. For example in a sprained ankle there is loss of control of locomotion. Even months after repair of ligamentous and capsular tissues has taken place, perception may still be distorted.

Structures containing connective tissue

Synovial joints (Fig. 3.7)

In synovial articulations, the bones involved are linked by a fibrous capsule, usually containing intrinsic ligamentous thickenings, and often also internal or external accessory ligaments. The articulating bony surfaces are generally not in direct continuity but are covered by hyaline articular cartilage of varying thickness and precise topology. Smooth movement of the opposing articular surfaces is aided by a viscous synovial fluid, which acts as a lubricant, and whose production requires the presence of a synovial membrane which is one of the defining characteristics of the joint type.

Fig 3.7 Example of a synovial joint.

Fibrous capsule and ligaments

In synovial articulations the bones are linked by a fibrous capsule of parallel and interlacing connective tissue fibres – a cuff that encloses the joint cavity. With some exceptions, each end is attached in a continuous line around the articular ends of the bones concerned. Within this the capsule is lined by synovial membrane. A fibrous capsule usually exhibits local thickenings of parallel bundles of collagen fibres, called capsular (intrinsic) ligaments, that are named by their attachments. Some capsules are reinforced by tendons of nearby muscles or expansions from them. Accessory ligaments are separate from capsules and may be extracapsular or intracapsular in position.

All ligaments are slightly elastic: collagen comprises about 70–80% of the dry weight, elastin 3–5%. They are taut at the normal limit of a particular movement but do not resist normal actions, since they are designed to check excessive or abnormal movements. Further they are also protected from excessive tension by reflex contraction of appropriate muscles.

The mechanical response of a ligament to a load can be represented on a load–deformation curve (Fig. 3.8). In the first part of such a curve (its foot) the ground substance is almost completely responsible for absorbing the stress and displaces the fibres in the direction of the stress. When the load is increased, ligamentous tissue responds slowly and maximum resistance to distraction is only possible if there is enough time for realignment of the collagen bundle. The linear part of the curve shows the slow elastic stretching of the collagen. During this stage, recovery of the original shape of the tissue occurs when the deforming load is removed. This slow rate of deformation is known as ‘creep deformation’. Even in this linear part of the curve, breaking of intermolecular crosslinks begins. For this reason it is assumed that, in physiological circumstances, the load on ligaments is kept within that shown in the foot of the curve, where collagen is not yet under undue strain and the role of ground substance is maximal.¹⁰ The composition and the amount of gel ground substance are therefore important in load bearing. On reaching the yield point, a non-elastic or plastic deformation occurs and the ligament progressively ruptures. Some investigators have found that in bone–ligament–bone preparations, separation occurs at the point of insertion.¹¹

Fig 3.8 Mechanical response of the anterior cruciate ligament of the knee to a load. 1. Foot of the curve, the ground substance alone almost completely absorbs the stress. 2. Linear part of the curve, slow elastic stretching of the collagen which is known as ‘creep deformation’. 3. Yield point, a non-elastic or plastic deformation occurs. 4 and 5, the ligament progressively ruptures. Redrawn from Frankel ³³ with permission.

In normal circumstances, mechanical stress induces early firing of mechanoreceptors in capsuloligamentous tissues. This causes a well-balanced reflex action of all musculotendinous units acting across the joint to avoid inert tissue becoming overloaded and damaged. If this muscular defence fails, strain falls on the ligament which is unable to stabilize the joint and so ruptures.

Synovial membrane and fluid

The synovial membrane lines the non-articular parts of synovial joints such as the fibrous capsule and the intra-articular ligaments and tendons within the margins of articular cartilage. The internal surface of the membrane has a few small synovial villi which increase in size and number with age. It also has flexible folds, fringes and fat pads. These accommodate to movement so as to occupy potential spaces and may promote the distribution of synovial fluid over the joint surfaces (Fig. 3.9).

Fig 3.9 A section of a synovial joint and its associated highly vascular synovial membrane in a human fetal hand. The two articular cartilage surfaces (A, arrowed) are separated on the right by a layer of synovial fluid (S) secreted by the synovial membrane (SM) which extends a short distance into the joint space from the capsule (C). From Standring, Gray’s Anatomy, 40^th edn. Churchill Livingstone, Edinburgh, 2008 with permission.

Structurally the membrane consists of a cellular intima which is one to four cells deep that rests upon a loose connective tissue subintima and contains the vascular and lymphatic network which has an important function in the supply and removal of fluid. On ultrastructural examination, two cell types (A and B) are apparent. These are closely involved not only with the production of synovial fluid ¹² but also in the absorption and removal of debris from the joint cavity. The A cells especially have marked phagocytic potential.¹³ Some synovial cells can also stimulate the immune response by presenting antigens to lymphocytes if foreign material threatens the joint cavity.¹⁴

Synovial fluid is a clear, viscid (glairy) substance formed as a dialysate containing some protein. It occurs not only in synovial joints but also in bursae and tendon sheaths. Secretion and absorption are functions of the cells of the intima and of the vascular and lymphatic plexus in the subintima. The synovial initima cells also secrete hyaluronan molecules into the fluid and much evidence has accumulated to show that the viscoelastic and plastic properties of the fluid are largely determined by its hyaluronan content. Chains of hyaluronan bind proteins; these complexes are negatively charged and in turn bind water. The biophysical process is similar to that of the proteoglycans in the matrix of connective tissue and a thick viscous liquid which resembles egg white is formed. Its viscosity varies widely according to circumstances. With a low rate of shear, water is driven out of the hyaluronan–protein complexes and the fluid becomes highly viscous; increase in shear lowers viscosity and the fluid tends to behave more like water. In contrast to viscosity, elasticity increases with higher rates of shear. Both viscosity and elasticity decrease with increasing pH and temperature.¹⁵

Cartilage

Articular cartilage is essentially a specialized type of connective tissue.

Composition

Although the same three tissue elements – cells, ground substance and fibres – are present, their properties differ from ordinary connective tissue and determine its biochemical and biomechanical behaviour.

The composition of proteoglycans in the ground substance changes with increasing depth

In the superficial layer, chrondroitin sulphate is a prominent constituent in the GAGs but the deeper layers contain more and more keratan sulphate. A high concentration of chrondroitin sulphate stimulates the condensation of thin collagen fibres to form a dense network at the surface but keratan sulphate enhances synthesis of thick, easily movable fibres in the deeper layers. This adapted synthesis influences the local architecture and strength and resistance to compressing and shearing forces.

Cartilage cells or chondrocytes occupy small spaces in the matrix

They are involved in the production and turnover both of type II collagen and ground substance, processes stimulated by variation in load.

Chondrocytes change with increasing depth from the surface 1,16 (Fig. 3.10). In the superficial stratum (zone 1), cells are small, flattened and disposed parallel to the surface. They are surrounded by fine tangentially arranged collagen fibres. A thin superficial layer of this zone has been shown to be cell-free. Cell metabolism in this part is low, which is consistent with the absence of wear and tear in normal healthy tissue. The cells of the intermediate stratum (zone 2) are larger and more rounded and those in the radiate stratum (zone 3) are large, rounded and arranged in columns perpendicular to the surface. In these deeper zones, cells are screened from the coarse fibres by a coat of pericellular matrix bordered by a network of fine collagen fibres. In this way, cells are protected against the stresses generated by load conditions.

Fig 3.10 Zones in articular cartilage. Zone 1, cells, small and flattened, disposed parallel to the surface; zone 2, cells become larger and more rounded; zone 3, cells are largest and arranged in columns, perpendicular to the surface; zone 4, mineralized cartilage. The border between mineralized and non-mineralized cartilage is called the ‘tide mark’.

The collagen fibres vary in structure and position with increasing depth from the surface (Fig. 3.11a)

In the superficial or tangential stratum, a dense network of fine fibrils is arranged tangential to the articular surface to resist tensile forces that result from compression on certain points of the articulating surface during normal activities. Analysis has also shown the existence of certain ‘tension trajectories’ in accordance with the more or less fixed patterns of tensile forces that take place during movements. These preferential directions have been elaborated during growth as a result of forces acting on the joint. Near the border of the joint, the fibrils blend with the periosteum and joint capsule.

Fig 3.11 (a) Arrangement of collagen fibres in articular cartilage. (b) and (c) Functioning of collagen fibres in articular cartilage. (b) non-load condition, (c) during load they are stretched in a direction perpendicular to the direction of force.

In the intermediate stratum, collagen fibres are coarser and more spread out to pursue an oblique course that forms a three-dimensional network. In non-load conditions the fibres are orientated at random but when load is applied they are immediately stretched in a direction perpendicular to that of the applied force (Fig. 3.11c). When the load is removed the fibres return to their original oblique position. This behaviour partly explains the resilience and elasticity of cartilagenous tissue.

In the radiate stratum, collagen fibres are arranged radially and correspond with the fibrous architecture of the subchondral and osseous lamina. The result is a series of arcades which extend from the deepest zone towards the surface.

Characteristics of cartilage

These include low metabolic and turnover rates, rigidity, high tensile strength, and resistance to compressing and shearing forces while some resilience and elasticity is retained. The proportion of collagen in matrix increases with age.

On the basis of variations in the matrix and the number of fibres present, cartilage in the locomotor system is divided into two types: hyaline and fibroelastic.

Most cartilage is hyaline: exceptions are the surfaces of the sternoclavicular and acromioclavicular joints and of the temporomandibular joints, all of which are of dense fibrous tissue. Although the light microscope appearance of hyaline cartilage is translucent, electron microscopy shows a system of fine fibrils and fibres. The water content is up to 80%. Strength, resistance and elasticity are the results of the proteoglycans in the ground substance together with the specific properties of the collagen fibres. Negatively charged proteoglycans bind a large number of water molecules and causes the cartilage to swell. Swelling is limited, however, by the increasing tension of the collagen fibre networks in the superficial and deep layers which are closely interconnected. The result is an elastic buffer that, together with the elasticity of the periarticular structures, dissipates the effect of acute compressive forces. It also provides the articular mechanism with some degree of flexibility, particularly at the extremes of range. If the load is applied over a very short time, cartilage deforms in an ‘elastic’ way almost without disturbance of its water content. However, if compression is maintained for hours, water is displaced to surrounding regions that are under less or no compression and the compressed cartilage undergoes ‘plastic’ deformation. In engineering terms, this slow predictable rate of deformation is known as ‘creep’ and is greatest within the first hour of compression. When the deforming load is removed, recovery of the original shape of the tissue occurs at a rate that is specific for each form of cartilage.

Water transport during dynamic load conditions probably also has significance in the transport of nutrients and metabolites to and from the chondrocytes.

Articular cartilage lacks a nerve supply and is also completely avascular. Nutrition is derived from three sources: synovial fluid, vessels of the synovial membrane and vessels in the underlying marrow cavity which penetrate the deepest part of the cartilage over a short distance. This last source is available only during growth because, after growth is completed, the matrix at the deepest part of the cartilage becomes impregnated with hydroxyapatite crystals which form a zone of calcified cartilage impenetrable by blood or lymph vessels (Fig. 3.10, zone 4).

Articular discs and menisci consist of fibroelastic cartilage and are predominantly fibrous. They separate certain articular surfaces that have a low degree of congruity (e.g. the knee and the radiocarpal joint). Their functional roles are to improve the fit between joint surfaces, distribute weight over a larger surface, absorb impacts and spread lubricant.

With age, articular cartilage becomes firmer but also thinner and more brittle. The number of cells decreases. In normal healthy joints these changes are extremely slow. Erosion particularly occurs when joints become dehydrated or when synovial fluid viscosity permanently alters. Replacement of an eroded surface by proliferation of deeper layers has not been demonstrated. Deposits of calcification and surface ruptures are signs of degeneration. Except in young children, regeneration cannot be expected. However, there is evidence that a defect can be filled with newly synthesized collagen.

Synovial bursae

In situations where skin, tendons, muscles or ligaments move in relation to other structures under conditions that involve fluctuating pressure, synovial bursae are formed to reduce friction. They can be compared with flattened sacs of synovial membrane which create discontinuity between tissues and provide complete freedom of movement over a short distance. A capillary film of synovial fluid on their internal surfaces acts as a lubricant. Depending on their position they are classified as subcutaneous, subtendinous, submuscular or subfascial bursae. Sometimes they communicate with the joint cavity with which their synovial membranes are continuous.

Nerves

Peripheral nerves also possess supporting connective tissue. Within the nerve trunk the efferent and afferent axons are grouped together in a number of fasciculi (Fig. 3.12). The bundled axons in the fasciculi lie roughly parallel, surrounded by loose delicate collagen fibres running longitudinally along them. Both structures show a wavy appearance which disappears when gentle traction is applied.

Fig 3.12 Connective tissue in peripheral nerves: epineurium, a collagen coat that encases the nerve trunk; perineurium, fine collagen and laminae of fibroblasts that surround each fasciculus; endoneurium, loose delicate collagen that surround axons in the fasciculi.

Each fasciculus is surrounded by a fibrous perineurium, a regular structure of flattened laminae of fibroblasts alternating with fine collagen, running in various directions. These fibroblasts are connected together and form a diffusion barrier against noxious chemical products, bacteria and viruses. In this way, the enclosed axons are to some extent isolated from the external environment. Inside this perineural tube a protein-poor liquid flows centrifugally. This axoplasma is cerebrospinal fluid, which is re-assimilated into the blood circulation at the end of the peripheral nerve. In this respect, the spinal canal and the endoneural spaces are continuous (Fig. 3.13).

Fig 3.13 Transverse section through a human peripheral nerve, showing the arrangement of its connective tissue sheaths. Individual axons, myelinated and unmyelinated, are arranged in a small fascicle bounded by a perineurium. P, perineurium; Ep, epineurium; E, endoneurium. Courtesy of Professor Susan Standring, GKT School of Medicine, London, in Standring, Gray’s Anatomy, 40^th edn. Churchill Livingstone, Edinburgh, 2008 with permission.

The epineurium encases the nerve trunk as a collagen coat with little regular organization. Connective tissue surrounding nerves serves as an important mechanical protection to maintain the conductile properties of the nerve.¹⁷ During movement, nerves are potentially exposed to tensile forces that can be avoided by mobility in relation to surrounding structures. Here, the wavy form of both axons and surrounding collagen fibres is an important consideration: this ‘waviness’ of the axons is paramount, allowing them to remain relaxed even when the collagen fibres are stretched. Thus, within the normal range of movement, the axons will be protected by the tensile force of the collagen component. When there is a severe sprain or fracture perhaps with dislocation, the range of plastic deformation of collagen can be exceeded and ultimately rupture of collagen fibres and neurotmesis results.

The tolerance of nerves to tension is much greater than it is to compression. However, the mobility of nerves allows them to move laterally, so avoiding a compressive force. When space is inadequate for such movement or the nerve is firmly anchored – which is the case in cervical nerve roots – the epineurium may absorb a certain amount of pressure but sooner or later the blood supply within the nerve is affected by increasing compression. Further compression may result in interference with the conductile properties of the nerve. In such circumstances, the Schwann cells and subsequently the myelinated sheath are damaged. Although the axons remain intact, action potentials become blocked, leading to loss of sensory and motor function (see Ch. 2).

Muscles

Muscular tissue consists of specialized cells or myofibrils embedded in a network of fine connective tissue that transmits the pull of the muscle cells during contraction to the adjacent parts of the skeleton. For this purpose, connections exist between the muscle cells and the finest collagen fibres of the connective tissue network.

The muscle cell or myofibril consists of sarcomeres or myofilaments – the basic contractile units of a muscle – arranged in parallel (Fig. 3.14). In each sarcomere two types of filament are distinguishable, chemically characterized as actin and myosin. The actin filaments are each attached at one end to the inner side of the cell membrane forming the so-called Z-line. At the other end they are free and interdigitate with the central myosin filaments. During muscle contraction, the actin filaments slide in relation to the myosin towards the centre of the sarcomere which brings the attachments at the Z-lines closer together with shortening of the whole contractile unit (Fig. 3.15).