Chapter 280 – CONNECTIVE TISSUE STRUCTURE AND FUNCTION

Connective tissue forms the architectural framework of the musculoskeletal system and serves as a scaffold that supports various organs and tissues. Many important structures within the body are composed primarily of connective tissue ( Table 280-1 ). For example, articular cartilage is made up exclusively of a specialized dense connective tissue without blood vessels, nerves, or lymphatics. Tendons, ligaments, intervertebral discs, and fascia are structures also consisting of dense connective tissue, although of a different composition from cartilage. Bone contains both dense and loose connective tissue; the latter provides support for bone marrow cells and helps to fill the space within the bone cavity. Adipose tissue is also often considered to be a specialized connective tissue that has both metabolic and structural functions.

Articular joints are formed by several common types of connective tissue, including muscle, bone, tendon, ligament, cartilage, meniscus, and synovium, which function as a unit during joint movement. These tissues demonstrate distinct characteristics when imaged by magnetic resonance because of their unique cellular and matrix composition ( Fig. 280-1 ). In this type of image, tissues with a greater water content, such as adipose tissue and cartilage, appear lighter than very dense tissues, such as the subchondral bone, which appear dark.

Despite their diversity, what connective tissues have in common is that they contain cells, usually of mesodermal origin, that have an abundant extracellular matrix. The cells within a particular connective tissue control the composition of the extracellular matrix, which, in turn, determines the physical properties of the tissue. The many types of connective tissue cells range from fibroblasts, which are present in the connective tissue found in skin, synovium, tendons, ligaments, and fascia, to more specialized cells such as chondrocytes and osteoblasts, which are found in cartilage and bone, respectively (see Table 280-1 ). Basement membranes are a more special form of connective tissue in that they are produced by epithelial and endothelial cells, rather than by mesenchymal cells. The connective tissue matrix consists of an intricate mixture of proteins and other macromolecules, which are responsible for the unique function of each particular type of connective tissue ( Fig. 280-2 ). Connective tissues that must withstand high tensile loads, such as tendons, have a high content of collagen fibers, whereas tissues that must resist compression, such as cartilage, have a high content of proteoglycans.

Because of the wide distribution of connective tissues within the human body, diseases that affect connective tissue cells or extracellular matrix proteins often have systemic effects. These connective tissue disorders can be divided into three general types: those that arise from a mutation within a gene encoding a connective tissue protein (e.g., Marfan syndrome); those that are the result of an inflammatory, sometimes autoimmune, process centered in connective tissues (e.g., rheumatoid arthritis); and those that are the result of a degenerative type of process often associated with aging (e.g., osteoarthritis). A basic understanding of the composition of the various connective tissues and of the way in which this composition relates to structure and function is important for understanding the clinical presentation, as well as management, of the spectrum of musculoskeletal and connective tissue disorders.

TABLE 280-1   -- DIVERSITY OF CONNECTIVE TISSUE–CONTAINING STRUCTURES

Structure Function Cells Key Matrix Components
Adipose tissue Energy storage, organprotection Adipocytes, fibroblasts Fine reticular fibers
Basement membrane Support and filtration barrier Epithelial and endothelial cells Type IV collagen, laminin
Bone Skeletal support, blood cell production, mineral storage Osteoblasts, osteoclasts, osteocytes Type I collagen, osteocalcin, bone sialoprotein, hydroxyapatite
Cartilage Allows joint motion, transmits loads Chondrocytes Type II collagen, aggrecan
Dermis Support and resiliency Fibroblasts Type I collagen, elastin
Ligament Connects bone to bone Fibroblasts Type I collagen, small proteoglycans
Tendon Connects muscle to bone Fibroblasts Type I collagen, small proteoglycans
Stroma Support of organs Fibroblasts, organ specific cells Type I, VI collagen, fibronectin
Synovium Joint lining, produces synovial fluid Fibroblasts, macrophages Type I, III collagen
Vessel wall Structural support of blood vessels Vascular smooth muscle cells Type III collagen, elastin

Magnetic resonance image of a knee joint demonstrating the diversity of connective tissue structures

FIGURE 280-1  Magnetic resonance image of a knee joint demonstrating the diversity of connective tissue structures. The image was obtained with a 1.5 Tesla magnet using a fast spin-echo T2-weighted imaging protocol. Tissues with a higher water content, such as adipose and articular cartilage, appear lighter, as does the synovial fluid. The subchondral bone is dark because of a high mineral and low water content and can be seen just below the very thin layer of articular cartilage.  (Courtesy of Dr. Carol Muehleman, Rush Medical College, Chicago.)

Model of extracellular matrix proteins and their interaction with each other and with matrix receptors on a chondrocyte.

FIGURE 280-2  Model of extracellular matrix proteins and their interaction with each other and with matrix receptors on a chondrocyte. Decorin, fibromodulin, and types IX and XI collagen all interact with type II collagen and regulate collagen fiber assembly and structure. Types II and VI collagen bind matrix receptors called integrins. Type II collagen can also bind to anchorin CII, whereas fibronectin binds to integrins. A large proteoglycan aggregate forms when multiple aggrecan molecules bind to a long strand of hyaluronic acid, which, in turn, is anchored to the cell by CD44. Additional matrix proteins shown include thrombospondin and cartilage oligomeric protein (COMP).  (Adapted from Knudson W, Loeser RF: CD44 and integrin matrix receptors participate in cartilage homeostasis. Cell Mol Life Sci 2002;59:36-44.)

CONNECTIVE TISSUE PROTEINS AND MACROMOLECULES

Each different type of connective tissue has a unique composition based on the amount and specific forms of extracellular matrix proteins and macromolecules. The extracellular matrix components can be grouped into families that include the collagens, proteoglycans, elastins, and other noncollagenous glycoproteins. These extracellular matrix components not only provide the scaffolding that forms the tissue, but also, importantly, create an information-rich environment that provides external cues to the connective tissue cells. Intracellular signaling, initiated through the interaction of extracellular matrix components with cell surface receptors, regulates certain important processes, including cell growth, differentiation, and survival and remodeling of the matrix. The integrins, named for their ability to integrate the extracellular matrix with intracellular cytoskeletal components, are a large and important family of cell surface receptors that recognize and bind some different extracellular matrix proteins initiating intracellular signaling (see Fig. 280-2 ). The signals generated by extracellular matrix proteins are integrated with signals generated by soluble mediators, also present in the matrix, including growth factors and cytokines. In normally functioning connective tissues, the cells, extracellular matrix proteins, and soluble mediators all work beautifully in concert to maintain the tissue and to adapt the tissue to meet changes in physical or biomechanical demands.

Collagens

Collagen is a major component of the extracellular matrix and is the most abundant protein in the body; it accounts for about 25 to 30% of the total protein mass. Collagens are triple-helical proteins formed when three polypeptide chains, called α chains, wind around each other to form a collagen molecule ( Fig. 280-3 ). The α chains are rich in the amino acids proline and glycine, which are important in forming the helical conformation of the polypeptide chain. Because of its ring structure, proline stabilizes the helix. Glycine, the smallest of the amino acids, is spaced at every third position in the chain such that it occupies the tightly spaced inside portions of the triple helix. Mutations that result in the substitution of a much larger amino acid for glycine can severely disrupt the triple helical structure. Post-translational modifications, including hydroxylation of specific proline and lysine residues, occur during the processing of the collagen molecules; these modifications are important in stabilizing the collagen structure and in forming interchain and intrachain cross-links.

 Collagen structure

FIGURE 280-3  Collagen structure. Three α chains (in this case, two procollagen α1 chains and one procollagen α2 chain) containing a high content of the amino acids glycine (Gly) and proline (Pro) wind together to form a triple helical collagen molecule (A). After secretion from the cell, the N-terminal and C-terminal ends are removed by proteases to form a collagen fibril monomer (B). Monomers are assembled to form a microfibril, which, in turn, is assembled into fibrils; finally, multiple fibrils form a complete collagen fiber (C).

More than 30 collagen genes code for the collagen α chains. α chains specific for each collagen type combine to form at least 19 different collagens. The types of collagen can be grouped by structure and function ( Table 280-2 ). In the fibrillar collagens (collagen types I, II, III, V, and XI), individual collagen molecules are packed in a staggered and ordered fashion to form a collagen fibril, which has a characteristic banding pattern when observed by high-power microscopy. Fibril formation occurs outside the cell after the N-terminal and C-terminal propeptides present on procollagen molecules are proteolytically removed to form mature collagen. The fibrils, in turn, are packed together to form fibers. Further cross-linking of collagen fibrils strengthens the structure. These rather stiff, ropelike structures provide the tensile strength for connective tissues. The fibril-associated collagens (collagen types IX, XII, XIV) help to form and stabilize the collagen fibrils. Unlike the fibrillar collagens, this group of collagens contains regions where the triple helical structure is interrupted, and this can result in a bend in the collagen molecule, hence the name fibril-associated collagens with interrupted triple helixes. Type IV collagen is the classic basement membrane collagen that forms in aggregates to provide structural support to the basement membrane.

TABLE 280-2   -- CONNECTIVE TISSUE COLLAGENS

Collagen Type Chains Molecules Tissue Distribution Selected Diseases from Mutations
FIBRILLAR COLLAGENS
I α1(I) [α1(I)]2α2(I) Skin, bone, tendon, organ capsules, arteries Osteogenesis imperfecta, Ehlers-Danlos type VIIA
  α2(I) [α1(I)]3
II α1(II) [α1(II)]3 Cartilage, vitreous humor Stickler's syndrome, osteoarthritis
III α1(III) [α1(III)]3 Skin, vessels, uterus Ehlers-Danlos type IV, aortic aneurysms
V α1(V) [α1(V)]3 Skin, vessels, placenta, chorion, uterus  
  α2(V) [α1(V)]2α2(V)    
  α3(V) α1(V)α2(V)α3(V)    
XI α1(XI), α2(XI) α1(XI)α2(XI)α1(II) Cartilage  
BASEMENT MEMBRANE COLLAGENS
IV α1-α5(IV) [α1(IV)]2α2(IV) Basement membranes Alport's syndrome, porencephaly
FIBRIL-ASSOCIATED COLLAGENS WITH INTERRUPTED TRIPLE HELIXES (FACIT)
IX α1-α3(IX) α1(IX)α2(IX)α3(IX) Cartilage, intervertebral discs Degenerative disc disease, osteoarthritis
XII α1(XII) [α1(XII)]3 Tendons, ligaments, other soft tissues  
XIV α1(XIV)   Ubiquitous  
NETWORK-FORMING COLLAGENS
VIII α1(VIII),α2(VIII)   Cornea, vessels  
X α1(X) [α1(X)]3 Growth plate cartilage Spondylometaphyseal dysplasia
LONG-CHAIN, ANCHORING-FIBRIL COLLAGEN WITH INTERRUPTED TRIPLE HELIX
VII α1(VII) [α1(VII)]3 Anchoring fibrils Epidermolysis bullosa
OTHERS
VI α1-α3(VI) α1(VI)α2(VI)α3(VI) Cartilage, stroma  
XIII α1(XIII)   Skin, gut  

The type and amount of collagen found in a particular connective tissue are important contributors to tissue function. Tissues such as bone, tendon, and cartilage, which must withstand high biomechanical loads, are packed with large amounts of long fibrillar collagens, primarily type I collagen in bone and tendons and type II collagen in cartilage. The loose connective tissues, such as basement membranes and organ stroma, experience much less mechanical force but require a matrix that allows more rapid diffusion of molecules and so contain a higher content of aggregating or anchoring types of collagen and less fibrillar collagen. Recent work correlating mutations in particular collagen genes with disease phenotypes in humans and studies of phenotype after gene disruption in transgenic mice have added to our knowledge of collagen function (see Table 280-2 ). Some examples include “brittle-bone disease” or osteogenesis imperfecta resulting from type I collagen mutations, severe premature osteoarthritis from type II collagen mutations, and fragile blood vessel and visceral walls in Ehlers-Danlos syndrome type IV from mutations in type III collagen. Excess collagen production can also disrupt tissue function, usually by causing tissue fibrosis as seen in scleroderma. Finally, fragile connective tissues can result from inadequate collagen production and cross-linking, as occurs in scurvy from vitamin C deficiency. Vitamin C (ascorbic acid) serves as a cofactor for the hydroxylation reaction necessary to modify specific prolyl and lysyl residues in procollagen and a deficiency in ascorbic acid reduces collagen secretion, stability, and cross-linking.

Proteoglycans

These matrix macromolecules consist of a protein core to which short oligosaccharides and longer chains of glycosaminoglycans are covalently attached. The large variety of proteoglycans is based on the types and lengths of glycosaminoglycans as well as the sequence and length of the protein core. Chondroitin sulfate, keratan sulfate, heparan sulfate, and dermatan sulfate are four different forms of glycosaminoglycans that consist of repeating disaccharide units. The sulfates present on the glycosaminoglycans create a highly negatively charged environment that is hydrophilic. Thus, connective tissues that contain large amounts of proteoglycans also contain relatively large amounts of water bound to the proteoglycans. In addition to water, proteoglycans bind cationic proteins, which, in some cases include growth factors resulting in a mechanism by which tissues can store growth factors in the matrix.

Aggrecan is a very large (molecular weight >200,000 kD) proteoglycan found in cartilage that contains a protein core of more than 2000 amino acids to which are attached approximately 100 long chondroitin sulfate chains, approximately 30 shorter keratan sulfate chains, and approximately 50 short oligosaccharides. Decorin, biglycan, fibromodulin, and lumican are examples of small proteoglycans that have much shorter protein cores (30 to 60 kD) and fewer glycosaminoglycan side chains. These small proteoglycans interact with collagen and function to regulate the formation of collagen fibrils and help to stabilize the collagen network (see Fig. 280-2 ). Although their functions are not completely understood, they also likely have other roles because of their ability to bind matrix molecules, in addition to collagen, and as a result of their affinity for growth factors.

Hyaluronic acid is a nonsulfated glycosaminoglycan that is synthesized in very long linear polymer strands. Hyaluronic acid is produced by chondrocytes and by synovial cells and is a major component contributing to the viscosity of the synovial fluid. Unlike the other glycosaminoglycans, hyaluronic acid is not attached to a protein core and so is not directly used to form proteoglycans. In cartilage, however, strands of hyaluronic acid bind aggrecan molecules through a non-covalent interaction with a globular portion of the N-terminus of the aggrecan core protein and a second small protein called link protein. The resulting macromolecular complex containing 100 or more aggrecan molecules linked to each long strand of hyaluronic acid forms a gel-like substance in cartilage as a result of its high sugar and water content.

The proteoglycan aggregates provide articular cartilage with resiliency. When cartilage is compressed during joint loading, water is pushed out from the proteoglycans, and the negative charges on the glycosaminoglycans become closer; this helps to resist further compression through charge repulsion. When the load is released, water is drawn back to the proteoglycans, and the result is fluid flow responsible for part of the biomechanical stimulation of the cells. The fluid flow also helps to move nutrients through the tissue, a feature that is particularly important for articular cartilage, a connective tissue that depends on diffusion from the synovial fluid for most of its nutrition. The network of collagen fibers that surrounds aggrecan prevents the proteoglycans from swelling beyond a certain size, thus creating a swelling pressure in cartilage that also helps to resist compression.

Noncollagenous Fibrillar Proteins

Elastic fibers are composed of elastin and proteins, often referred to as microfibrils, such as fibrillin, because their fibrils are much smaller than the classic collagen fibrils. Elastic fibers must withstand repeated stretching and deformation and must be capable of returning to a relaxed state. Tropoelastin is the precursor to elastin and is synthesized by several cell types, including vascular smooth muscle cells and dermal fibroblasts, where it contributes to the elasticity of the blood vessel wall and skin, respectively. Elastin also plays a similar role in the lung. Elastin, like collagen, forms interchain and intrachain cross-links that help to stabilize and strengthen the elastic fibers, although the stiffness of the elastin fibers is less than that of the collagen fibers. Many of the cross-links in elastin are formed through the hydroxylation of two unique amino acids found in tropoelastin, desmosine and isodesmosine. Fibrillin is found in several elastic tissues and is particularly abundant in the aorta, the suspensory ligament of the lens, and the periosteum. Fibrillin was a little-known protein until investigators discovered that fibrillin mutations are the cause of most cases of Marfan's syndrome.

Noncollagenous Glycoproteins

Along with the proteoglycans, the many other noncollagenous matrix glycoproteins found in connective tissues form much of what is sometimes described as the ground substance in histologic terms. This rather inert-sounding description should in no way be taken to suggest that these matrix components simply fill in or hold the tissue together. Rather, these proteins participate actively in creating the information-rich environment described earlier. Proteins in this group include fibronectin, vitronectin, osteopontin, laminin, and thrombospondin. Fibronectin is found in most connective tissues throughout the body. Laminin is particularly prominent in basement membranes, and osteopontin is found in greater amounts in cartilage and bone. All these proteins bind to cells through specific cell surface receptors to promote attachment of cells to the matrix. The proteins also interact with other matrix proteins, such as collagen and proteoglycans, to integrate the cells further with the extracellular matrix. Through the interactions with cell receptors and other matrix proteins, the noncollagenous glycoproteins function in regulating tissue morphogenesis as well as in tissue repair and remodeling. They also appear to play a role in other diverse processes, including tumor growth and metastasis.

Other Matrix Proteins

Many other matrix proteins present in connective tissues, such as tenascin, osteonectin, the matrilins, matrix Gla protein, and osteocalcin, are less completely understood but are probably no less important. Matrix Gla protein and osteocalcin are both found in bone, whereas matrix Gla protein is also present in cartilage and other soft tissues, including blood vessel walls. Both proteins require reduced vitamin K as a cofactor for a post-translational carboxylation reaction important to the cation binding properties of the proteins. Inhibition of vitamin K reduction by the drug warfarin interferes with this reaction, as it does for the coagulation factors. Warfarin is contraindicated in pregnancy because it has been shown to cause embryopathy, which includes abnormalities in skeletal formation likely secondary to effects on matrix Gla protein and/or osteocalcin.

Extracellular Matrix Protein Receptors

Connective tissue cells use several different types of cell surface receptors to bind extracellular matrix proteins. The major types of receptors include integrins, CD44, discoidin domain receptors (DDRs), and proteoglycan family receptors such as the syndecans ( Table 280-3 ). CD44 is expressed by several different connective tissue cell types as well as by nonconnective tissue cells such as lymphocytes. On connective tissue cells, CD44 is the principal receptor for hyaluronan. CD44 binding of hyaluronan is particularly important in forming a gel-like pericellular coat found around certain connective tissue cells such as chondrocytes. DDR1 and DDR2 are receptor tyrosine kinases found on epithelial and mesenchymal cells. DDR1 can bind types I through VI and type VIII collagen, whereas DDR2 is activated only by fibrillar collagens. Syndecans contain a transmembrane protein core to which heparan sulfate proteoglycans are attached to the extracellular domain. The proteoglycans bind growth factors such as fibroblast growth factor. Syndecans also appear to interact with integrins and may modulate integrin function.

TABLE 280-3   -- CELL-SURFACE RECEPTORS FOR EXTRACELLULAR MATRIX COMPONENTS

Receptor Ligands
CD 44 Hyaluronan
Syndecan (1–4) Tenascin, fibronectin, fibroblast growth factor (FGF)
Discoidin domain receptors 1, 2 Collagen
Integrins  
 α1β1 Collagen, laminin, cartilage matrix protein
 α2β1 Collagen, laminin, chondroadherin
 α3β1 Laminin, fibronectin, collagen, epiligrin
 α4β1 Fibronectin, VCAM-1
 α5β1 Fibronectin
 α6β1 Laminin
 α7β1 Laminin
 α8β1 Tenascin, nephronectin, fibronectin, vitronectin
 α9β1 Laminin, collagen, tenascin
 α10β1 Collagen
 αvβ1 Fibronectin, vitronectin
 αvβ3 Vitronectin, fibrinogen, osteopontin, fibronectin, thrombospondin
 αvβ5 Vitronectin, fibronectin
 αvβ6 Fibronectin, tenascin
 αvβ8 Vitronectin, laminin, collagen, fibronectin

The integrins represent the largest family of cell surface matrix receptors and have been found to be the primary receptors for many matrix proteins, including collagens, fibronectin, laminin, vitronectin, osteopontin, and thrombospondin (see Table 280-3 ). Integrins are heterodimeric transmembrane proteins consisting of one α and one β subunit. More than 20 known integrins are formed by 14 types of α subunits and at least nine types of β subunits. The two major subfamilies of integrins function in mediating interactions between cells and extracellular matrix proteins. These are the β1 subfamily and the αv subfamily. The specificity of extracellular matrix protein binding is determined by both the α and the β subunit, although more than one integrin type can bind to the same matrix protein, often at different sites within the protein. For example, the α1β1, α2β1, α3β1, and α10β1 integrins are all capable of binding collagen, whereas α3β1, α4β1, α5β1, and αvβ3 integrins can all bind fibronectin. In addition, each type of integrin can bind more than one different type of extracellular matrix protein; for example, αvβ3 can bind vitronectin, osteopontin, or fibronectin.

Each connective tissue cell expresses a combination of integrins that is regulated, at least in part, by the mix of proteins present in the extracellular matrix. In addition, integrin expression and affinity for matrix ligands can change in response to cues from the matrix, including stimulation by growth factors and cytokines. The exact function of each particular integrin is currently a topic of investigation by numerous research laboratories. Research in bone has found that the αvβ3 integrin present on osteoclasts plays an important role in the ability of these cells to resorb bone. For this reason, chemical inhibitors of αvβ3 are being tested in early clinical trials for the treatment of osteoporosis.

Binding of extracellular matrix proteins to integrins activates certain signal transduction pathways that regulate cell responses to the matrix, including changes in gene expression. Signaling complexes are formed at sites, often referred to as focal adhesions, where close contact is made between the integrins and the extracellular matrix. The signaling complexes include activation of several different pathways involving tyrosine and serine-threonine kinases, phosphoinositide, and arachidonic acid, as well inducing ion fluxes. Accompanying, and intimately tied to, activation of cell signaling are changes in the organization of the cytoskeleton. In this way, integrins can mediate processes that require changes in cytoarchitecture such as cell migration and wound repair. Integrins also provide signals necessary to promote cell survival in cells that require attachment to a matrix to survive. Given the diverse roles of integrins in mediating events important to connective tissue development, repair, and remodeling, studies of integrin function should provide important new information needed to understand connective tissue in health and disease.