Connective Tissue: Tissue in Action by Elzi Volk

Athletes are mostly concerned with increasing strength or speed in specific activities. Increasing muscle size and power, endurance abilities, fuel utilization efficiency: these are often the primary concerns in training. We spend much time, effort and money in maximizing our muscle capabilities. However, an integral part of our anatomy often takes a back seat: connective tissue.

Soreness from exercising is a familiar experience, often an accepted incidental result of training. Most soreness results from muscle tissue trauma, but stress is also induced upon the tissues connected to the muscles: bones, tendons and ligaments. These tissues are also subject to a manifestation of the second law of thermodynamics: aging.

The human body is a magnificent piece of machinery with rubber bands, hinges and joints throughout. Unlike the tin man, the oilcan is not a option when our joints become ‘rusty’. When Humpty Dumpty had his great fall, all the king’s army couldn’t put him back together again. But if the tin man and poor Humpty had taken proper care of their connective tissue, perhaps they would have healed and continued squatting in their ripe old age.

Aside from muscle tissue, connective tissue trauma is a major source of physical discomfort, especially in athletes. This is not surprising, considering that connective tissue is one of the most abundant and widely distributed tissues in the body. It forms our bones, surrounds our organs, holds our teeth in place, cushions and lubricates our joints, and connects the muscles to our skeleton.

Connective tissue serves multiple functions. It provides the organism with shape and mechanical support. Also, it modulates cell migration, growth, and differentiation. The following are major features of connective tissue:

Structural and mechanical: Connective tissue supports the shape of cells, tissues, and organs interacting with the cytoskeleton. The most obvious structural connective tissue is bone, comprising the skeleton and supporting the entire organism.

Defense: It contains cells and metabolites important in immune function, such as inflammation, and in tissue repair after injury.

Nutrition and transport of molecules: Blood vessels, a connective tissue, transports substances throughout the body. Components in connective tissue regulate movement of nutrients between cells.

Storage: Adipose tissue is a unique connective tissue, providing storage of energy and insulation.

Connective tissue function is mediated by its different components, most of which are macromolecules that interact with one another and with the cells. Varying the proportions and the arrangement of the individual components determine the function of the particular tissue. Several diseases are due to known defects in the primary structure of one or more components. Disruptions in the regulatory mechanisms of degradation contribute to conditions such as osteoarthritis and complications in diabetes. As well, nutrient deficiencies and various drugs may disrupt regulation of tissue synthesis and degradation.

This article examines the influences of various factors on the connective tissue types most affected by athletics: cartilage, tendons, and ligaments. Information on the histology and physiology of connective tissue is presented to enable readers to understand how nutrition and various pharmaceuticals influence the synthesis, degradation and remodeling of these tissues. Along with a brief discussion of the effects of specific activities and metabolic disorders, the roles of nutrition and several common drugs are examined.

Connective tissue is metabolically active and serves many structural functions. The tissue consists of three basic components: fibers, ground substance and cells. The fibers and ground substance, which exist outside the cells, are collectively called the extracellular matrix. The matrix separates and protects cells, bearing weight, tension and offering defense. Mixed populations of cells with various functions interact with the extracellular matrix contributing to the various mechanisms in tissue physiology. Except for cartilage, connective tissue is innervated (has a nerve supply). Vascularity (blood supply) in tendons and ligaments is sparse, whereas cartilage is avascular (has no blood supply). Further examination of the various components will elucidate their contributions toward these attributes.

Fibers: Three major types of fibrous proteins appear in connective tissue: collagen, elastic, and reticular fibers. Tissues contain one or more types of collagen, and different proportions of collagen and elastic fibers. Fibers associate with additional components of the extracellular components and minerals to form specialized connective tissue. The predominant fiber type usually determines a tissue’s specific properties. Expression of connective tissue types is largely a function of inherent cell types, external environment, and physiological age of the organism.

Collagen is the most abundant protein, comprising of ~30% of total protein in the body. At least 14 types of collagen have been isolated and characterized, and most tissues contain several types. Collagen types may form fibrils or sheets, providing strength or elasticity. All collagens share a common structure, varying in their chemical composition, macromolecule organization, tissue distribution and function. The most common collagen types are described in Table 1. The other types are considered minor, though they are found in nearly all tissues in tiny amounts.

Synthesis of collagen involves a cascade of biochemical modifications of the original building blocks. Many enzymes, cofactors and growth promoters influence these modifications which are crucial to the structure and function of mature collagen. The most abundant form of collagen, Type I, consists of three units of polypeptide chains, which are comprised of subunits of amino acids. Differences in the chemical structure of the polypeptide chains determine the different collagen types. These chains intertwine in a triple helix to form molecules called procollagen. Procollagen is formed within the cell and subsequently transported outside of the cell into the extracellular matrix. Outside the cell, procollagen is altered to form tropocollagen, which is then able to form into microfibrils. Microfibrils form fibrils when they are packed together in an overlapping fashion. The microfibrils are held by hydrogen bonds, hydrophobic interactions and reinforced by cross-links between tropocollagen molecules. The characteristic types and amounts of their cross-links largely determine the mechanical properties of collagen fibers. As we shall see throughout this article, nutrient deficiencies and pharmaceuticals may influence synthesis or metabolism during the critical stages in development of collagen.

In collagen Type I and III, fibrils form fibers. These fibers associate to form bundles in Type I collagen, but fibers are not formed in Type II collagen (cartilage). The diameter of the fibers depends on the number of fibrils they contain. Changes in fibril diameter are often seen in aging and influenced by some pharmaceuticals such as anabolic steroids.

Patterns between collagen types and orientation differ between various connective tissues and even species. In general, collagen fibers are inelastic, but have great tensile strength giving a combination of flexibility and strength to the tissues they are in. The diameter of the fibers and their orientation along the lines of stress constitutes the tensile strength of a tissue.

Elastic fibers predominate in tissues subject to stretching, such as ligaments, but are also found with collagen in tendons, arteries, and skin. The length, thickness and distribution of elastic fibers differ in the various tissues. Elastic fibers are composed of elastin enclosed within tubular microfibrils, and are thinner and tauter than collagen fibers. These fibers stretch easily, up to one and one-half times their original length when the deforming force is relaxed. Each elastin fiber forms a cross-linked network with other elastin. The entire network can expand and contract like a rubber band.

Reticular fibers are extremely thin and branch to form an extensive network in certain organs such as in smooth muscle, adipose tissue, and bone marrow. These fibers consist of collagen protein associated with glycoproteins and proteoglycans. During inflammation and wound healing, most connective tissues have abundant reticular fibers, which are subsequently replaced by regular collagen fibers.

Ground substance: The ground substance fills the spaces in-between the cells and fibers. Its viscosity acts as a lubricant due to the high water content. Soluble precursors of the fibrous proteins, proteoglycans, glycoproteins and other molecules secreted by cells are abundant in the ground substance.

The two major components of the ground substance are the proteoglycans and structural glycoproteins, which trap water molecules and lend strength, rigidity and resiliency to the extracellular matrix. The chemical structure of proteoglycans determines their size and structural function and are examined further.

Proteoglycans are large molecules formed by many linear chains of polysaccharide units called glycosaminoglycans (GAGs). Proteoglycan monomers are grouped according to the length and type of GAG chains attached to a protein core. These GAG chains radiate out from the core like bristles of a bottlebrush. Six classes of GAGs consist of repeating modified two-sugar subunits. An amino sugar (glucosamine or galactosamine) is always present as one of the two sugars in the repeated subunits. These subunits are modified by the addition of sulfate groups. As we will see, sulfation of the GAGs determines their biological activity. Proteoglycan monomers may combine further with a chain of hyaluronic acid, an unsulfated GAG, to form larger proteoglycan complexes. As we shall see, these complexes, which may contain hundreds of attached proteoglycan aggregates, constitute a significant role in cartilage tissue.

Proteoglycans act as a molecular sieve moderating the movement of cells, and nutritive and inflammatory substances. The are also responsible for attracting and maintaining water balance within the tissue. The long chains of GAGs are negatively charged due to the carboxylic (COO-) and sulfate (SO₄-) groups of the amino sugars. The high density of negative charges attracts and binds water molecules.

Depending on the structure and types of GAGs, proteoglycans can trap as much as 50 times their weight in water. The hyaluronic-proteoglycan aggregate molecules in cartilage resemble long centipedes with hairy legs. The negatively charged GAGs ("hairs") repel each other and give the complex an open structure, which occupies a lot of space. The highly polar 'hairs' attract water molecules and the complex acts like a stiff sponge bounded by the collagen network. When this 'sponge' is compressed, some of the bound water is lost and therefore absorbs forces and redistributes them equally. This is how cartilage protects structures in the joint from mechanical (stress and weight) damage.

Glycoproteins are similar to proteoglycans. In contrast, the protein fraction predominates over the carbohydrates, which are branched structures. The primary glycoproteins are fibronectin, laminin, and chondronectin. Their role in the ground substance is the migration and adhesion of cells to their substrates. For example, fibronectin connects fibroblasts to collagen and binds to proteoglycans. These compounds contribute to the scaffolding in connective tissue to provide support and influence movement of cells.

Cells: The various cells in connective tissue store vital metabolites and synthesize fibrous proteins and other components of the extracellular matrix. They play important roles in immune and inflammatory responses as well as in tissue repair. Many cells are indigenous to connective tissue. Some originate in bone marrow, but are constantly present in the tissue. These resident cells and their functions are listed in Table 2. Other cells migrate from blood vessels in response to tissue injury, inflammation and repair. They tend to disappear as healing progresses and inflammation subsides. These cells include plasma cells, neutrophils, monocytes and basophils. The discussion of inflammation will portray their role in tissue repair.

Each class of tissues contains a fundamental cell type that exists in mature and immature form. The active cells that proliferate and secrete the ground substance and fibers are indicated by the suffix -blast (meaning "forming"). The primary blast cells are fibroblasts, chondroblasts and osteoblasts (see Table 2). Once the adult matrix is formed, the blast cells assume a less active and mature mode and are indicated with the suffix -cyte (meaning a cell). However, when the tissue is injured, cells can revert to their more active mode to regenerate and repair the matrix components.

The rate of secretion of different substances by the same cell varies with the age and hormonal influences of the organism. Individual cells, such as the fibroblasts in connective tissue, rarely divide into new cells unless the tissue requires additional cells as when a tissue is damaged. During inflammation and repair, the numbers of fibroblasts increase within some connective tissues. The extracellular matrix greatly influences function and differentiation of the cells. Tensile forces may also influence cell function, as changes in cell shape may alter the responsiveness of the cell to hormones and growth factors (3). The discussions on inflammation and pharmaceuticals examine the influences of hormones and growth factors on cell function.

To understand how connective tissue is influenced by activities, nutrition and pharmaceuticals, we must understand some basic physiology of these tissues. Synthesis and degradation of tissues is a continual process and are integral parts of tissue remodeling and turnover. Many modulators can affect these processes, as we will see in the ensuing discussion.

Each of the intracellular and extracellular events involved in macromolecule synthesis is subject to alteration or biochemical modification. Changes in gene transcription and in events after translation of macromolecules can alter distribution and deposition of tissue proteins and proteoglycans. For example, many pathological conditions are attributable to abnormal or insufficient collagen synthesis, such as scurvy and vitamin C deficiency.

Several modulators regulate synthesis and degradation of connective tissue components: enzymes and cofactors, hormones and growth factors, and cytokines. Because their roles are so complex, examination of these specific regulators is beyond the scope of this article. However, energy and nutritional status and various drugs influence them and they will be discussed where relevant in the ensuing context.

Degradation of tissue components occurs during growth, remodeling, inflammation, and repair of tissues. It can take place during any point in synthesis of the various components. For example, scurvy is a disease caused by deficiency of dietary vitamin C. Ascorbic acid (vitamin C) is required in the enzymatic hydroxylation of the prolyl and lysyl residues of collagen. Procollagen molecules lacking the hydroxyproline residues have an unstable triple helix formation, are susceptible to alteration and they are inadequately cross-linked. Consequently, these molecules are then mechanically unstable and prone to degradation. Therefore, the degree of degradation occurs at a much greater rate than synthesis of collagen fibers. This is more pronounced in area where collagen renewal tales place at a fast rate.

Specific enzymes initiate degradation of macromolecules. Collagenases, which degrade collagen fibers, are synthesized by various cell types and stimulated by hormones, prostaglandins, and other substances secreted by lymphocytes and macrophages. Metal ions, such as calcium, also regulate collagenase activity. Enzymes within cellular lysosomes degrade proteoglycans. We will see how synthesis and degradation are an integral part of tissue turnover.

Remodeling of tissues is the process of changing and replacing tissue components with others. Normal remodeling during growth or repair requires a proper balance of synthesis and degradation of tissue components. Proteoglycans in the extracellular matrix appear to regulate remodeling of connective tissue by influencing collagen formation during the repair process. Remodeling is also regulated by mechanical stimulation. Mechanical tension and compression modify bone and cartilage remodeling, where tissues such as these depend on diffusion of nutrients for maintenance since they have no direct blood supply.

Turnover of connective tissue is the net balance between synthesis and degradation of the macromolecules. A turnover negative balance is characteristic of several inflammatory and joint diseases where degradation occurs at a higher rate than synthesis. The previous example of scurvy and ascorbic acid deficiency portrays the significance of turnover balance of tissue components. The repair process in tissue injury involves managing macromolecule turnover so that synthesis equals degradation.

Turnover rate of various connective tissue components varies. Elastin may take months to years for renewal. Collagen is also a stable protein and renewal is slow. Replacement of mature collagen can require weeks to several months. Collagen turnover rates vary in different structures. Tendon collagen renewal is very slow, whereas the collagen of loose connective tissue that surrounds our organs is renewed more rapidly.

Many alterations of connective tissue metabolism may be due to changes in cross-linking in both collagen and elastin fibers. Differences in amino acid composition of cross-links influence the stability and turnover of these fibers. Although the same enzymes process the collagen types and elastin during synthesis, the resultant cross-links may be significantly different. Some types of cross-links are more stable than others and therefore indirectly influence turnover of fibers. For instance, 50-80% of collagens from tissues in bone and cartilage is glycosylated and has a relatively higher turnover rate. However, collagen fibers in tendons are not glycosylated and have a lower turnover rate.

Proteoglycans turnover rapidly: 2-4 days for hyaluronic acid and 7-10 days for the sulfated proteoglycans. In adult humans, 250 mg of proteoglycans are catabolized in one day (5). The polysaccharide chains of the proteoglycans are subject to modifications similar to those of collagen

Injury to connective tissue involves damage to the cells and structural components of the tissue. Several responses are triggered and a sequence of events begins to repair the tissue. The reaction to injury includes vascular, cellular and biochemical responses which are outlined here.

Three phases of the repair process can be applied to the general healing of connective tissue (7). These phases, however, may overlap. These responses prevent the spread of damaging agents to nearby tissues, dispose of damaged cells, and replace damaged tissue with newly synthesized components.

Acute inflammation phase: Immediately after injury, several vascular and cellular reactions initiate the response known as inflammation. The process begins with a release of chemical mediators from cells into the extracellular fluid. The initial tissue damage stimulates release of histamine from mast cells, which causes dilation of blood vessels in the local area and increases vascular permeability. Increased blood flow and fluids and proteins that leak from the permeable blood vessels cause edema in the tissue and consequent swelling. Cells migrate from nearby blood vessels and cause release of more inflammatory mediators, such as kinins and prostaglandins (PGs). Local tissue pressure and some of these mediators act on nearby nerves to cause pain. These events lead to the classical signs of inflammation: redness, swelling, pain and heat.

The primary purpose of inflammation is to rid the site of damaged tissue cells and set the stage for tissue repair. Acute inflammation generally lasts from 48 to 72 hours after an injury and gradually subsides as the repair process progresses. Many of the events that occur during this time initiate tissue repair. PGs are considered important mediators of inflammation and are often the target of intervention with anti-inflammatory agents. However, PGs may also have a significant role in tissue repair. Many immigrant cells also have significant roles in tissue remodeling. Leukocytes (white blood cells), such as neutrophils and monocytes, accumulate within the damaged tissue along with resident macrophages. Enzymes released from these cells help digest necrotic cells and degrade matrix molecules; neutrophils and macrophages engulf cell debris. Blood platelets release growth factors that stimulate new fiber and matrix molecule synthesis.

Matrix and cellular proliferation phase: Chemical mediators released by inflammatory cells stimulate migration and proliferation of fibroblasts, which participate in the repair process. Fibroblasts secrete fibronectin, proteoglycans and small diameter Type III collagen fibers. In addition to these fibers, newly formed capillary channels, clotting proteins, platelets and freshly synthesized matrix molecules form granulation tissue. However, this granulation tissue has little tensile strength.

Remodeling phase: Recall that remodeling reshapes and strengthen damaged tissue by removing and reforming the matrix and replacing cells. As repair progresses, inflammatory cells disappear, the number of blood vessels and the density of fibroblasts decrease. The proportion of Type I collagen to Type III collagen and the matrix organization increases. Collagen fibers are reoriented in the direction of loading, especially in ligament repair. Collagen matures and elastin forms; tensile strength increases. However, the remodeled tissue may not completely resemble the original and thus the mechanical capabilities of that tissue may be altered.

Now that we have a background in connective tissue biology, we will commence Part II with discussion of specific connective tissues and some of their pathophysiology. We will also examine the roles of nutrition and several pharmaceuticals in tissue healing and remodeling.

Reference

1. Fawcett, DW. Textbook of Histology, eleventh edition. 1986. W.B. Saunder, PA.

2. Goldberg B, Rabinovitch M. Connective tissue. In. Cell and Tissue Biology, sixth edition, Weiss, L, ed. 1989:158-188. Urban & Schwarzenberg, Baltimore.

3. Schnaper HW, Kleinman HK. Regulation of cell function by extracellular matrix. Pediatr Nephrol 1993, 7:96-104.

4. Smith CA, Wood EJ. Cell Biology, 1994. Chapman and Hall, London.

5. Paroli E, Antnilli L, Biffoni M. A pharmacological approach to glycosaminoglycans. Drugs Exptl Clin Res 1991, 17:9-20.

6. Oakes, BW. The classification of injuries and mechanisms of injury, repair and healing. In: Textbook of Science and Medicine in Sport. Bloomfield J, Fricker PA, Fitch KD, eds. 1991:200-217.Human Kinetics Books, Ill.

7. Alvarez OM, Uitto J, Perejda AJ, eds. Pharmacological and environmental modulation of Wound Healing. In: Connective Tissue Disease, Molecular Pathology of the Extracellular Matrix, vol. 12 The Biochemistry of Disease 1987:367-383. Dekker, NY.

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Connective Tissue Part 1: Tissue in Action

Reference