Connective tissue

The structure of connective tissue was covered to some extent in the course on The Human Body: structure and function. Connective tissue consists of cells surrounded by a matrix of molecules which supports the tissue cells. The matrix molecules are mainly proteins and carbohydrates, together with some salts. Within the matrix is the extra-cellular fluid which carries the nutrients to the cell, and the cellular wastes back to the blood or the lymph. It is through this fluid that all exchanges between the cell and the rest of the body must take place.

The matrix proteins are particularly rich in the amino acid, glycine, which accounts for about one amino acid residue in every three. The other amino acids which predominate in the connective tissue matrix proteins are serine, proline, threonine, and alanine. All connective tissue proteins in all species of animals show this same preponderance of these five amino acids. That is, the connective tissue proteins in the animal kingdom have a characteristic structure.

The connective tissue matrix is not just an inert support system for cells, and a medium for exchange. It also influences cell migration and development, and in particular it is needed for the repair of damaged tissue. This area is considered in detail later in this module.

The connective tissue matrix contains five principal elements: collagens, elastic fibres, proteoglycans, structural glycoproteins, and basement membranes.

Collagens are structural proteins rich in the amino acids hydroxyproline and hydroxylysine, with a glycine residue at every third position on the amino acid chain. There are several different types of collagen molecules which are found in the different tissues, each with characteristic properties. For example, the collagen found in uterine tissue is different from that found in bone, or in cartilage. And even during a process such as wound healing where collagen fibres are produced, different types of collagen fibres are formed at different stages.

Collagen synthesis, like any protein synthesis, is directed by a sequence of bases on a DNA molecule, but for the synthesis to occur there must be hydroxylation of the amino acids proline and lysine, a set of reactions which requires vitamin C. If there is a deficiency of vitamin C, wound healing is impaired (figure 11•1).

Even in normal tissue there is constant replacement of the collagen fibres, requiring their catabolism and synthesis. This turnover of molecules is characteristic of all tissues in the body. While the rate of turnover of collagen is slow, it demands a means for the regulation of its levels in the organs and tissues where it is found. This means there must be a balance maintained between collagen synthesis and collagen catabolism. The enzymes responsible for collagen catabolism, and therefore for its turnover, are the collagenases, secreted mainly by fibroblast cells, but also by macrophage cells and endothelial cells (figure 11•1). There are regulators of collagenase activity which therefore influence collagen turnover.

In those tissues such as the uterus, lung, skin, and blood vessels where elasticity is needed as well as strength, the connective tissue contains a considerable proportion of elastic fibres. The presence of these fibres allows these tissues to be distorted, and still return to their original shape. These fibres contain protein whose structure is similar to that of collagen, although they lack the hydroxylated amino acids.

Proteoglycans, as their name suggests, are composed mainly of carbohydrate and protein, arranged as a protein core around which are polymers of carbohydrate molecules. These molecules, which readily take up water to form a gel, are found in most biological fluids, particularly in connective tissues and at the surface of cell membranes. The nature of these molecules differs between tissue types. For example, in cartilage the proteoglycans contain considerable amounts of chondroitin sulphate. Proteoglycans in dermal connective tissue contain hyaluronic acid in abundance, while in those within the basement membranes there is a preponderance of heparitin sulphate.

Collagen could be classed as a structural glycoprotein. It is not the only such molecule, for there is a range of glycoproteins, molecules which are combinations of protein and carbohydrate, which provide a degree of rigidity to tissues and organs. The fibronectin group of molecules are examples of these. They are found in two main areas: blood plasma, and connective tissue. Plasma fibronectin is a soluble form of this molecule, whose main role is to bind to other molecules in the connective tissue, such as collagens and proteoglycans, as well as to cell surfaces, bacteria, and even DNA strands. This binding is important not only in attaching tissue cells to components of the connective tissue, but also in promoting the formation of a clot of fluid at the site of an injury, an important aspect of early wound healing. Plasma fibronectin is synthesised in the liver. Tissue fibronectin is synthesised by most cells of the body. This latter fibronectin serves similar functions to plasma fibronectin.

The final components of connective tissue to be considered are the basement membranes, fine structures which have sufficient strength to support layers of cells, and to provide a site for the attachment of the cells. The basement membranes in some tissues are important for their filtration properties, particularly in the glomeruli in the kidneys. There the basement membrane of these small capillaries acts with the endothelial cells to restrict the passage of molecules from the blood into the Bowman's capsule. Collagen is the main component of basement membranes, although a number of other glycoproteins are involved as well. Basement membranes are synthesised by the cells which are resting on them.

The structure of most tissues is dependent on a healthy, intact basement membrane supporting the cells. Any damage to the basement membrane usually has severe consequences for the function of the tissue. This is a point which will be emphasised many times during this course, for in many disease states the main cause of the symptoms can be traced back to some abnormality in a basement membrane.

Collagens, elastic fibres, proteoglycans, structural glycoproteins, and basement membranes are the important components of connective tissue structure and function. Each plays a role in tissue repair.

Tissue repair

The healing process requires two stages. Firstly, there is the removal of dead tissue and debris from the site of the injury or infection. Secondly, there is replacement of this dead tissue by living tissue. This is emphasised in figure 11•2.

As was explained in the previous module, the initial response at an inflammation site is a migration into the site of inflammatory cells, cells which secrete molecules to break down the tissue and attack foreign matter. As a part of this there is an accumulation of fibronectin and other proteins, platelets, and other substances which produce a closely woven net of molecules in the inflammation site. Such an aggregation of molecules and platelets helps to prevent fluid loss, and to protect the underlying tissues from further damage. This initial response is followed by a second invasion of cells called fibroblasts, a class of large cells which synthesise protein and collagen. There are several different types of fibroblasts, with some of them being capable of phagocytosis. While the fibroblast cells are responsible for much of the healing in the inflammation site, other cell types and a range of secretions also are involved in this process.

The removal of dead tissue and accumulated debris from the inflammation site is the task of the macrophage cells and the various enzymes they secrete. While this activity is taking place, the tissue repair commences. This is looked on as occurring in three phases: wound contraction, wound repair, and tissue regeneration. But it should not be thought that these activities are distinct from each other. In any inflammation site all three processes may be occurring simultaneously, together with the removal of debris.

These activities of tissue repair are summarised in figure 11•3.

The contraction phase of wound repair is the result of the action of specialised cells called myofibroblasts, whose activities resemble those of a cross between a smooth muscle cell and a fibroblast. Within two or three days after the injury, myofibroblasts appear at the injury site, where their numbers increase. They form attachments to the tissues within the injury, and then contract. This results in a general contraction of the injured tissue to the centre of the site, reducing the extent of the injury. In this way the amount of tissue replacement which is required is considerably reduced, generally by as much as two thirds. The smaller the area for tissue replacement, the faster is the healing. If this wound contraction does not occur then there is a greater chance for extensive formation of scar tissue which may be permanent. On the other hand, if the contraction is excessive this also can adversely affect the healing process.

Wound repair is the second phase of the healing process. It is during this phase that granulation tissue is formed, as was mentioned in the last module. This starts with the accumulation of fibroblast cells in the injury site about two or three days after the insult. These cells secrete many of the substances needed to renew the connective tissue at the site, including proteoglycans, fibronectin, and collagen. One of the functions of the proteoglycans is to take up fluid, which is why some wounds appear to have a degree of oedema. At the same time there is a stimulated growth of vascular tissue, the endothelial cells in particular. These cells, derived from existing endothelial cells at the periphery of the injury, eventually are formed into new capillaries which then make connections with the existing vascular bed. This is an important process since it restores the circulation to the repair site, ensuring that the very active cells at this site are supplied with their nutrients, and that the wastes are removed. In some instances the newly forming capillaries emerge from the surface of the repairing tissue to appear as small red granules, which is why this stage of tissue repair is referred to as granulation tissue. In repairing tissue there eventually is developed a network of capillaries which is far more extensive than that found in normal tissues.

An interesting aspect of wound healing is that there is a very rapid turnover of the collagen laid down at the wound site. This is a result of the activities of macrophage cells and fibroblasts in secreting the enzyme collagenase into the site. The reason for this unusually high turnover rate of collagen in the repairing tissue is not known, although it could allow for adjustment of the orientation of the collagen fibres in accordance with the stresses which are placed on the wound site. It could therefore allow for flexibility in the way in which the wound is repaired in accordance with the use of the tissue.

After approximately two or three months of wound repair there is an overall increase in the amount of collagen in the wound site. In addition, cross-links are developed between the collagen fibres to further increase their strength. At the same time some of the new blood vessels in the wound site are destroyed, which accounts for the change in the wound from a red colour to a pale colour as the scar is formed. The amount of blood flowing through the site determines its colour.

The final stage of the wound healing involves the replacement of the scar tissue by regenerated tissue from the surrounds of the injury site. For example, if the wound is in skin, the scar tissue forms a protective coat over the newly regenerating skin cells, and eventually is shed once the new skin has covered the site. This process of tissue regeneration is covered in the next section in this module.

In summary, tissue repair takes place in several phases and involves a range of cell types and substances. The first event in the repair, or at least the first event in reducing the extent of the damage, is the formation of a clot at the wound site. This clot and the chemicals associated with it attract macrophage cells and fibroblasts. These cells then promote the removal of debris, damaged tissue, and microorganisms from the injury site by the secretion of various enzymes, and by their phagocytic activity. The fibroblasts then secrete the components which form the basis of the new connective tissue at the wound site. At the same time the adjacent endothelial cells are stimulated to generate new blood vessels through the repairing tissue, forming granulation tissue. The strengthening of the connective tissue, together with a reduction of the blood supply, converts the repair tissue into a scar. Under the protection of a scar, tissue regeneration can occur.

Tissue regeneration

Wound repair mechanisms help to heal the wound site and allow the wound to be replaced with connective tissue. However, for the wound site to be restored to its previous function and structure, the process of regeneration must take place. This means that there must be a replacement of the connective tissue and scar tissue by the functional cells which were at that part of the body prior to the insult. For example, in the case of a skin injury, the tissue repair processes ensure that the wound is replaced with connective tissue and scar tissue. Regeneration is the process whereby the skin tissue is restored to the area.

Regeneration usually takes place by a proliferation of the surrounding tissue cells, as is indicated in figure 11•4. In the case of the skin, once again, this means that after the tissue repair there must be some regeneration of the epithelial cells surrounding the wound, with the newly formed skin cells invading the wound site. Gradually the epithelial cells derived from the surrounding skin cover the wound site and differentiate to form the different layers of skin, and to have the normal functions of skin.

Regeneration also involves some migration of cells. This requires the cells to detach from their basement membrane in the normal tissue surrounding the wound site, and then to move into the wound. There they may proliferate. The factors which stimulate the cells to migrate in this way, and which regulate their wanderings, are not known.

The process of differentiation, defined in figure 11•5, is important in regeneration, since a tissue usually consists of several related cell types derived from a more generalised parent cell. This is seen in a section through the skin, for example, where the lower cells of the epidermis divide to give rise to cells, which as they become closer to the skin surface take on different characteristics. In the process of regeneration this means that the cells which first invade the site must give rise to all of the other cells of that tissue, implying that they undergo some type of differentiation. In some tissues this may require the differentiation of cells into quite specialised forms. As a part of this process the original migrating cells may need to dedifferentiate, which means that they become more generalised in their function. Once aligned within the injury site they then can allow different parts of their DNA to be suppressed or expressed until they take on the functions of the specialised cells usually found at that site. Whatever the mechanism underlying this, differentiation of the regenerating cells is an important aspect of wound healing.

The factors which promote this regeneration of tissue are not fully understood. However, many different agents are known to be involved, some of which are listed in figure 11•6. These include the hormones insulin, glucagon, thyroxin, calcitonin, parathyroid hormone, and glucocorticoids. Fibronectin, a protein found in repairing tissue, and which is ubiquitous in the body since it is found in plasma and is secreted by most cells, also promotes regeneration. In addition to these factors are a number of specific growth factors which usually promote the regeneration of particular tissue types. Thus there is brain growth factor, epidermis growth factor, nerve growth factor, and a more general growth factor from the platelets called platelet-derived growth factor. While most of these agents are needed for aspects of the regeneration of all tissues, the specific requirements of each tissue vary. That is, the combination of growth promoters needed for liver regeneration is different from that required for epidermal regeneration.

This still leaves two questions about the regeneration of tissue cells. The first is the initial trigger for the cell proliferation. The second question concerns the regulation of the proliferating cells, the mechanism which stops them from constantly dividing. Once again the answers to these questions are unknown. Certainly it is known that many cells exhibit contact inhibition, which means that when they come into contact with other cells of their own type they stop replicating. But this is a crude regulatory mechanism which does not account for the three dimensional regeneration of a tissue such as liver. If more than about 70% of the mass of a liver is lost, this organ starts to regenerate the lost tissue at an amazing rate. When the liver has reached approximately the proportions of the original liver, the regeneration stops. One theory to account for this is that each liver cell secretes a minute amount of an inhibitor which is ineffective until a certain number of cells is secreting this inhibitor. But this area of the control of cell growth still is the subject of a considerable amount of research, not only because of its importance in tissue regeneration, but also because it is critical in understanding the growth of tumours.

Tissue regeneration, following tissue repair, is an important aspect of the healing of a wound or infection site. These two processes ensure that normal structure and function are restored to the damaged tissue.

Factors affecting healing

The tissue repair and regeneration mechanism which has just been described can be influenced by a number of factors, some of which improve the healing process, and some of which hinder it. Factors which assist wound healing are summarised in figure 11•7.

One of the more obvious influences on the outcome of any healing activity is the nature of the insult to the tissue, and the extent of the tissue damage. The smaller the wound the faster and more effective is its healing process. If the wound is clean this also speeds the healing since there is not the necessity to remove contaminating material. If there is an infection at the wound site this could cause excessive formation of granulation tissue, which results in a considerable amount of unwanted scarring.

The location of the wound is important. If the wound is in an area where there is an extensive blood supply, this speeds the healing. This is why a wound on the fingers heals more quickly than a wound on the heel, since finger skin is renewed more rapidly than that on the heel. People with poor circulation generally have a reduced rate of wound healing compared with people with a normal circulation. This is one of the reasons why leg ulcers, for example, often take a long time to heal in people with varicose veins. It also accounts for the slower healing of wounds in older people because of their generally reduced circulation.

Immobilising a wound enhances the repair process since it allows the connective tissue network to be established within the wound. If a wound frequently changes shape during the healing process the renewing blood vessels will not have an opportunity to make their appropriate connections. Also if the person participates in sport or other exercise this increases their circulating levels of glucocorticoid hormones, which are known to reduce the rate of wound repair. In general, adrenal steroid hormones inhibit wound repair by inhibiting the inflammatory response, and by reducing the rate of protein synthesis. Protein synthesis is essential for wound repair.

Sunlight helps to heal wounds because of the action of ultraviolet light.

Diabetics are at particular risk if they are injured, since their wounds are readily infected. This possibly is a consequence of the large amounts of glucose in their body fluids, with this glucose providing a nutrient support for a range of infective microorganisms and fungi. Any infection of a wound site impairs the healing process.

Several nutrients not only promote wound healing, but also are essential for this process. Vitamin C is needed for the formation of connective tissue, and for the restoration of a vascular supply to the wound site. Methionine, an essential amino acid, also is needed for healing. So too is zinc, a trace element which is a cofactor for several enzymes. This need for nutrients explains why wound healing quite often is ineffective in malnourished people.

Differences between tissues

An important aspect of wound healing is the tissue in which the injury is found. Different tissues have differing abilities to recover from an insult.

The skin is an example of a tissue which has extraordinarily good powers of recovery following an injury. The inflammatory response is rapid, and the wound healing effective. Tissue regeneration at the wound site in the skin ensures that the damaged skin is replaced, and over time little evidence usually remains to indicate that there was damage at that site. This means that the skin not only has a good ability to heal a wound, but also that it has good regenerative powers in its cells. Probably every person has had experience of this remarkable healing ability of their skin. But not all tissues have this ability to repair themselves so completely and so rapidly.

The liver is an example of a tissue which has an excellent regenerative capacity. If a majority of the liver is removed, then this organ is stimulated to undergo a massive and rapid regeneration until the bulk of the liver tissue is replaced. Even if a small amount of liver tissue is damaged, then the surrounding cells regenerate until the damage is repaired, and the restored area functions normally. However, if there is repeated injury to the liver, instead of there being regeneration of the hepatocytes there is excessive connective tissue formation. The result is an excessive amount of scar tissue development called fibrosis. This is one of the outcomes of cirrhosis of the liver. In effect this means that there is a reduced functional capacity of the liver.

The ability of lung tissue to regenerate and restore normal function to a damaged area depends on the location of the damage, and the extent of the damage. If the damage is confined to the lining of the air passages it is repaired quickly and effectively. However, if the damaging stimulus is persistent, as it generally is in the case of the toxic effects of cigarette smoke, the risk of tissue changes, particularly the formation of tumours, increases. Within the alveoli, damage by toxic fumes generally is repaired by tissue regeneration, allowing the repaired alveoli to function normally. This assumes that the damage is restricted to the alveolar cells, for if the damage is more extensive and affects the basement membranes supporting the alveoli, the repair processes result in scar tissue formation. In turn this reduces the effective area for gas exchange. This is one of the causes of emphysema, a shortness of breath which often is seen in smokers.

Kidney tissue, like lung tissue, also exhibits differing degrees of repair in response to insults. In general, the regenerative ability of the kidneys is poor, although there are differences between the various tissue types. The glomeruli have virtually no capacity to regenerate following damage, and any injury normally results only in their replacement by scar tissue. This reduces the functional capacity of the kidney. This is why diseases which affect the glomeruli, such as glomerulonephritis, can have long-term affects on the patient. The tubule cells of the kidney, particularly those in the renal cortex, readily regenerate to replace damaged cells, providing the damage is limited to the cells and the basement membrane is intact. If the basement membrane is damaged, replacement is less likely, and scar tissue formation is the likely outcome. In the medulla of the kidney the tubules have a reduced ability to regenerate, which means that damage in this area usually results in scar tissue formation. In some cases this scar tissue may impede flow through the tubules.

Myocardial cells in the heart cannot regenerate. Therefore, after a myocardial infarction, or following any infection of the myocardial cells, only scar tissue and damaged cells remain. For the heart this presence of damaged tissue means that it has a reduced muscular capacity. This not only is because of the reduced number of contractile cells, but also because these damaged cells reduce the effectiveness of the surviving cells.

Nerve cells in the central nervous system, like myocardial cells, have no ability to regenerate. This means that damaged cells in the brain or spinal cord are not replaced. However, if the cells are not completely destroyed they do have some limited ability to develop other axons which can make contact with surrounding neurons, and in this way restore some functions to the damaged brain or spinal cord. But this depends on the original neurons surviving the insult, as well as having the ability to make contact with other neurons. And in any case this attempted axonal renewal lasts for only a relatively short time after the original injury, usually only a week or two, after which scar tissue formation occurs. In the brain, scar tissue is formed when glial cells proliferate to occupy the space formerly held by neurons. That is, glial cell accumulation in damaged areas of the brain is the equivalent in the brain of scar tissue formation in the rest of the body. The glial cells do not have any of the functional aspects of the neurons they replace.

In summary then, the ability of different tissues to repair damage depends on the nature of the tissue and the nature of the damage. Most tissues which commonly are subjected to stresses of various types have considerable repair abilities. However, some vital tissues, such as nervous tissue and cardiac muscle, are unable to repair and restore damaged cells.