Connective Tissue Flashcards

Connective Tissue

Connective tissue provides support, connects tissues, and facilitates nutrient and waste exchange. Unlike epithelium, muscle, and nerve tissues, the major component of connective tissue is the extracellular matrix (ECM).

• Extracellular Matrix (ECM): Consists of protein fibers (collagen and elastic fibers) and ground substance.
• Ground Substance: Includes anionic, hydrophilic proteoglycans, glycosaminoglycans (GAGs), and multiadhesive glycoproteins (laminin, fibronectin). Glycoproteins stabilize the ECM by binding to other components and to integrins in cell membranes. Ground substance facilitates nutrient and waste exchange between cells and blood supply.

The diversity in connective tissue types arises from variations in cells, fibers, and ground substance. All connective tissues originate from embryonic mesenchyme, derived from the mesoderm.

• Mesenchyme: Viscous ground substance with few collagen fibers. Mesenchymal cells are undifferentiated with large nuclei and prominent nucleoli.
• Mesenchymal cells are often spindle-shaped with thin cytoplasmic processes. These cells migrate, surrounding and penetrating developing organs.
• Mesenchyme produces connective tissue proper and specialized connective tissues like bone and cartilage. It also includes stem cells for blood, vascular endothelium, and muscle.

Cells of Connective Tissue

Fibroblasts are key cells in connective tissue proper, originating from mesenchymal cells and residing permanently in the tissue. Other cells like macrophages, plasma cells, and mast cells come from hematopoietic stem cells in bone marrow and circulate in the blood before moving into connective tissue.

• Fibroblasts: Produce and maintain extracellular components. They synthesize collagen, elastin, GAGs, proteoglycans, and multiadhesive glycoproteins. ECM components undergo modification outside the cell before assembling as a matrix.
• Active Fibroblasts: Intense synthetic activity, abundant cytoplasm, much rough endoplasmic reticulum (RER), and a well-developed Golgi apparatus, with a large, ovoid, euchromatic nucleus and a prominent nucleolus.
• Quiescent Fibroblasts (Fibrocytes): Smaller, spindle-shaped, less RER, and a darker, more heterochromatic nucleus.
• Fibroblasts are targets of growth factors that influence cell growth and differentiation. They resume mitotic activity when tissue repair is needed.
• Myofibroblasts: Involved in wound healing, have contractile function, and are enriched with actin.

The regenerative capacity of connective tissue is evident in organs damaged by ischemia, inflammation, or trauma. Spaces left after injuries are filled by connective tissue, forming dense irregular scar tissue.

Wound healing depends on fibroblast activity. Myofibroblasts, with features of both fibroblasts and smooth muscle cells, are important for wound contraction.

• Adipocytes (Fat Cells): store lipid as neutral fats and insulate organs.
• Adipose connective tissue cushions and insulates the skin and other organs. Adipocytes have major metabolic significance.

Macrophages are phagocytic cells involved in turnover of protein fibers and removal of apoptotic cells and debris, especially at inflammation sites. Macrophage size and shape vary with activity.

• Macrophages: measure 10-30 μm in diameter and have an eccentrically located, oval or kidney-shaped nucleus.
• Referred to as "histiocytes" by pathologists.
• Macrophages are key components of the innate immune defense system. They remove cell debris, neoplastic cells, bacteria, and other invaders. Macrophages are also antigen-presenting cells required for lymphocyte activation.
• Activated Macrophages: Increased phagocytosis, intracellular digestion, metabolic activity, and lysosomal enzyme activity. They secrete enzymes for ECM breakdown and growth factors or cytokines that regulate immune cells and reparative functions.
• Stimulated macrophages can fuse to form multinuclear giant cells, usually found in pathologic conditions.

Macrophages have an irregular surface with pleats, protrusions, and indentations, related to pinocytotic and phagocytic activities. They have well-developed Golgi complexes and many lysosomes. Macrophages derive from monocytes circulating in the blood.

• Mononuclear Phagocyte System: Monocytes cross the epithelial wall of small venules to enter connective tissue, where they differentiate into macrophages. Many macrophage-like cells in various organs have specialized names. All are long-living cells that become activated during inflammation and tissue repair. Macrophages secrete growth factors and function in antigen presentation for lymphocyte activation.

Mast cells are oval or irregularly shaped cells, 7-20 μm in diameter, filled with basophilic secretory granules that often obscure the nucleus. Granules are electron dense and of variable size, ranging from 0.3 to 2.0 μm in diameter

• Mast cell granules display metachromasia due to acidic radicals in sulfated GAGs, changing the color of basic dyes from blue to purple or red. Granules are poorly preserved by common fixatives.
• Mast cells release bioactive substances important in local inflammatory response, innate immunity, and tissue repair.
• Molecules Released:
  • Heparin: Anticoagulant.
  • Histamine: Increases vascular permeability and smooth muscle contraction.
  • Serine proteases: Activate mediators of inflammation.
  • Eosinophil and neutrophil chemotactic factors: Attract leukocytes.
  • Cytokines: Direct activities of leukocytes and immune cells.
  • Phospholipid precursors: Converted to prostaglandins, leukotrienes, and lipid mediators of inflammation.
• Mast cells are numerous near small blood vessels in skin and mesenteries (perivascular mast cells) and in digestive and respiratory tracts (mucosal mast cells).
• Release of chemical mediators from mast cells promotes immediate hypersensitivity reactions. Anaphylactic shock is a dramatic example of immediate hypersensitivity reaction. The first exposure to an antigen causes antibody-producing cells to produce IgE that binds to mast cells. Upon a second exposure, the antigen reacts with IgE, triggering rapid release of histamine, leukotrienes, chemokines, and heparin.

Degranulation of mast cells also occurs due to complement molecules. Mast cells originate from progenitor cells in the bone marrow, circulate in the blood, cross venules, and enter connective tissues, where they differentiate.

Plasma cells are lymphocyte-derived, antibody-producing cells with basophilic cytoplasm rich in RER and a large Golgi apparatus near the nucleus. The nucleus is generally spherical but eccentrically placed. Plasma cells are present in most connective tissues and have an average life span of 10-20 days.

• Plasma cells are derived from B lymphocytes that are responsible for the synthesis of immunoglobulin antibodies.
• Plasma cells react only with the one antigen that stimulated the clone of B cells
• Bound antigen-antibody complexes are quickly removed from tissues by phagocytosis

Leukocytes (white blood cells) are wandering cells in connective tissue that leave the blood by migrating between endothelial cells of venules. This process increases during inflammation, a defensive response to injury or foreign substances. Inflammation begins with the local release of chemical mediators that induce events characteristic of inflammation: increased blood flow and vascular permeability, entry and migration of leukocytes, and activation of macrophages for phagocytosis.

• Most leukocytes function in connective tissue for a few hours or days and then undergo apoptosis.

Increased vascular permeability is caused by vasoactive substances such as histamine released from mast cells. The major signs of inflamed tissues include “redness and swelling with heat and pain” (rubor et tumor cum calore et dolore).

Chemotaxis draws leukocytes into inflamed tissues.

Fibers

The fibrous components of connective tissue are elongated structures formed from proteins that polymerize after secretion from fibroblasts. The three main types of fibers include collagen, reticular, and elastic fibers.

Collagen

The collagens are a family of proteins that form extracellular fibers, sheets, and networks that are strong and resistant to shearing and tearing forces. Collagen is a key element of all connective tissues, epithelial basement membranes, and external laminae. Collagen comprises 30% of the body’s dry weight and is secreted by fibroblasts and other cell types. There are 28 types of collagens, categorized according to the structures formed by their interacting α-chains.

• Fibrillar Collagens (Types I, II, III, V, XI): aggregate to form large fibrils clearly visible in the electron or light microscope. Collagen type I is the most abundant and forms large, eosinophilic bundles called collagen fibers. They densely fill the connective tissue.
• Network-Forming Collagens (Type IV, X): subunits produced by epithelial cells and are major structural proteins of external laminae and all epithelial basal laminae.
• Linking/Anchoring Collagens (Type VII, IX, XII, XIV): short collagens that link fibrillar collagens to one another and to other components of the ECM. Type VII collagen binds type IV collagen and anchors the basal lamina to the underlying reticular lamina in basement membranes.

Collagen synthesis occurs in fibroblasts. The initial procollagen α chains are polypeptides made in the RER. Selected alpha chains are aligned, stabilized by disulfide bonds, and folded as a triple helix. The triple helix undergoes exocytosis and is cleaved to a rodlike procollagen molecule.

Keloids are local swellings caused by abnormally large amounts of collagen that form in scars of the skin.

Here are the steps for collagen preparation:

1. Procollagen α Chains Production: Produced on polyribosomes of the RER, these chains have long central domains rich in proline and lysine. In type I collagen, every third amino acid is glycine.
2. Hydroxylation: Hydroxylase enzymes in the ER cisternae add hydroxyl (-OH) groups to some prolines and lysines in reactions that require O_2, Fe^{2+}, and ascorbic acid (vitamin C) as cofactors.
3. Glycosylation: Glycosylation of some hydroxylysine residues occurs to different degrees in various collagen types.
4. Triple Helix Formation: The amino- and carboxyl-terminal sequences of alpha chains have globular structures that lack the Gly-X-Y repeats. In the RER, the C-terminal regions of three selected alpha chains are stabilized by cysteine disulfide bonds, which align the three polypeptides and facilitates their central domains folding as the triple helix. The trimeric procollagen molecule is transported through the Golgi apparatus, packaged in vesicles, and secreted.
5. Procollagen Peptidases Action: Outside the cell, specific proteases called procollagen peptidases remove the terminal globular peptides, converting the procollagen molecules to collagen molecules. These now self-assemble into polymeric collagen fibrils near the cell surface.
6. Association with Other Molecules: Certain proteoglycans and other collagens (e.g., types V and XII) associate with the new collagen fibrils, stabilize these assemblies, and promote the formation of larger fibers from the fibrils.
7. Cross-Linking: Fibrillar structure is reinforced and disassembly is prevented by the formation of covalent cross-links between the collagen molecules, a process catalyzed by lysyl oxidase.

Type I collagen fibrils have diameters ranging from 20 to 90 nm and can be several micrometers in length. Adjacent rodlike collagen subunits of the fibrils are staggered by 67 nm, with small gaps (lacunar regions) between their ends. This structure produces transverse striations with a regular periodicity visible by EM. Type I collagen fibrils assemble further to form large collagen fibers that may be further bundled by linking collagens and proteoglycans.

Collagen turnover and renewal in normal connective tissue is generally a very slow but ongoing process. Degradation is initiated by specific enzymes called collagenases, which are members of the matrix metalloproteinases (MMPs), which clip collagen fibrils or sheets so that they are susceptible to further degradation by nonspecific proteases. Various MMPs are secreted by macrophages and play an important role in remodeling the ECM during tissue repair

Normal collagen function depends on the expression of many different genes and adequate execution of several posttranslational events. Many pathologic conditions are directly attributable to insufficient or abnormal collagen synthesis

Reticular Fibers

Reticular fibers are found in delicate connective tissue of many organs, notably in the immune system, made of collagen type III, which forms an extensive network (reticulum) of thin (diameter 0.5-2 μm) fibers for the support of many different cells. Reticular fibers are seldom visible in hematoxylin and eosin (H&E) preparations but are characteristically stained black after impregnation with silver salts and are thus termed argyrophilic. Reticular fibers are also periodic acid–Schiff (PAS) positive, which, like argyrophilia, is due to the high content of sugar chains bound to type III collagen α chains. Reticular fibers contain up to 10% carbohydrate as opposed to 1% in most other collagen fibers.

Reticular fibers produced by fibroblasts occur in the reticular lamina of basement membranes and typically also surround adipocytes, smooth muscle and nerve fibers, and small blood vessels. Delicate reticular networks serve as the supportive stroma for the parenchymal secretory cells and rich microvasculature of the liver and endocrine glands. Abundant reticular fibers also characterize the stroma of hemopoietic tissue (bone marrow), the spleen, and lymph nodes where they support rapidly changing populations of proliferating cells and phagocytic cells.

Elastic Fibers

Elastic fibers are thinner than type I collagen fibers and form sparse networks interspersed with collagen bundles in many organs, particularly those subject to regular stretching or bending. Elastic fibers have rubberlike properties that allow tissue containing these fibers, such as the stroma of the lungs, to be stretched or distended and return to their original shape. In the wall of large blood vessels, especially arteries, elastin also occurs as fenestrated sheets called elastic lamellae. Elastic fibers and lamellae are not strongly acidophilic and stain poorly with H&E; they are stained more darkly than collagen with other stains such as orcein and aldehyde fuchsin.

Elastic fibers (and lamellae) are a composite of fibrillin (350 kDa), which forms a network of microfibrils. Both proteins are secreted from fibroblasts and smooth muscle cells in vascular walls and give rise to elastic fibers in a stepwise manner. Initially, microfibrils with diameters of 10 nm form from fibrillin and various glycoproteins. The microfibrils act as scaffolding upon which elastin is then deposited. Elastin accumulates around the microfibrils, eventually making up most of the elastic fiber, and is responsible for the rubberlike property. The elastic properties of these fibers and lamellae result from the structure of the elastin subunits and the unique cross-links holding them together.

Elastin molecules have many lysine-rich regions interspersed with hydrophobic domains rich in lysine and proline that are thought to form extensible, random-coil conformations. During deposition on the fibrillin microfibrils, lysyl oxidase converts the lysines’ amino groups to aldehydes and four oxidized lysines on neighboring elastin molecules then condense covalently as a desmosine ring, cross-linking the polypeptides. Bound firmly by many desmosine rings, but maintaining the rubberlike properties of their hydrophobic domains, elastic fibers stretch reversibly when force is applied. Elastin resists digestion by most proteases, but it is hydrolyzed by pancreatic elastase.

Fibrillins comprise a family of proteins involved in making the scaffolding necessary for the deposition of elastin. Mutations in the fibrillin genes result in Marfan syndrome, a disease characterized by a lack of resistance in tissues rich in elastic fibers. Patients with this disease often experience aortic swellings called aneurysms, which are life-threatening conditions.

Ground Substance

The ground substance of the ECM is a highly hydrated (with much bound water), transparent, complex mixture of three major kinds of macromolecules: glycosaminoglycans (GAGs), proteoglycans, and multiadhesive glycoproteins. Filling the space between cells and fibers in connective tissue, ground substance allows diffusion of small molecules and, because it is viscous, acts as both a lubricant and a barrier to the penetration of invaders. Physical properties of ground substance also profoundly influence various cellular activities.

When adequately fixed for histologic analysis, its components aggregate as fine, poorly resolved material that appears in TEM preparations as electron-dense filaments or granules.

• GAGs (Glycosaminoglycans): Also called mucopolysaccharides, GAGs are long polymers of repeating disaccharide units, usually a hexosamine and uronic acid. The hexosamine can be glucosamine or galactosamine, and the uronic acid can be glucuronate or iduronate. The largest and most ubiquitous GAG is hyaluronan (also called hyaluronate or hyaluronic acid). With a molecular weight from 100s to 1000s of kDa, hyaluronan is a very long polymer of the disaccharide, glucosamine-glucuronate. Hyaluronan forms a viscous, pericellular network that binds a considerable amount of water, giving it an important role in allowing molecular diffusion through connective tissue and in lubricating various organs and joints.
• All other GAGs are much smaller (10-40 kDa), sulfated, bound to proteins (as parts of proteoglycans), and are synthesized in Golgi complexes. The four major GAGs found in proteoglycans are dermatan sulfate, chondroitin sulfates, keratan sulfate, and heparan sulfate, all with different disaccharide units modified further with carboxyl and sulfate groups. Their high negative charge forces GAGs to an extended conformation and causes them to sequester cations as well as water. These features provide GAGs with space-filling, cushioning, and lubricant functions.
• Like glycoproteins, they are synthesized on RER, mature in the Golgi apparatus, where the GAG side chains are added, and secreted from cells by exocytosis. Unlike glycoproteins, proteoglycans have attached GAGs that often comprise a greater mass than the polypeptide core. After secretion, proteoglycans become bound to the hyaluronan by link proteins and their GAG side-chains associate further with collagen fibers and other ECM components
• Proteoglycans are distinguished by their diversity, core proteins, each with one or many sulfated GAGs of different lengths and composition. A region of ECM may contain several different. Perlecan is the key proteoglycan in all basal laminae. One of the best-studied proteoglycans, aggrecan, is very large (250 kDa), having a core protein heavily bound with chondroitin and keratan sulfate chains. A link protein joins aggrecan to hyaluronan. Abundant in cartilage, aggrecan–hyaluronan complexes fill the space between collagen fibers and cells and contribute greatly to the physical properties of this tissue. Other proteoglycans include decorin, with very few GAG side chains that bind the surface of type I collagen fibrils, and syndecan, with an integral membrane core protein providing an additional attachment of ECM to cell membranes.

Embryonic mesenchyme is very rich in hyaluronan and water, producing the characteristic wide spacing of cells and a matrix ideal for cell migrations and growth. In both developing and mature connective tissues, core proteins and GAGs (especially heparan sulfate) of many proteoglycans bind and sequester various growth factors and other signaling proteins. Degradation of such proteoglycans during the early phase of tissue repair releases these stored growth factors, which then help stimulate new cell growth and ECM synthesis.

The degradation of proteoglycans is carried out by several cell types and depends in part on the presence of several lysosomal enzymes. Several disorders have been described, including a deficiency in certain lysosomal enzymes that degrade specific GAGs, with the subsequent accumulation of these macromolecules in tissues. The lack of specific hydrolases in the lysosomes has been found to be the cause of several disorders, including the Hurler, Hunter, Sanfilippo, and Morquio syndromes.

Due to high viscosity, hyaluronan and proteoglycans tend to form a barrier against bacterial penetration of tissues. Bacteria producing hyaluronidase, reduces the viscosity of the connective tissue ground substance and have greater invasive power.

Making up the third major class of ground substance macromolecules, multiadhesive glycoproteins all have multiple binding sites for cell surface integrins and for other matrix macromolecules. The adhesive glycoproteins are large molecules with branched oligosaccharide chains and allow adhesion of cells to their substrate. An example is the large (200-400 kDa), trimeric glycoprotein laminin with binding sites for integrins, type IV collagen, and specific proteoglycans, providing adhesion for epithelial and other cells. All basal and external laminae are rich in laminin, which is essential for the assembly and maintenance of these structures.

Another glycoprotein, fibronectin, is a 235-270 kDa dimer synthesized largely by fibroblasts, with binding sites for collagens and certain GAGs, and forms insoluble fibrillar networks throughout connective tissue. The fibronectin substrate provides specific binding sites for integrins and is important both for cell adhesion and cellular migration through the ECM.

Integrins are integral membrane proteins that act as matrix receptors for specific sequences on laminin, fibronectin, some collagens, and certain other ECM proteins. Integrins bind their ECM ligands with relatively low affinity, allowing cells to explore their environment without losing attachment to it or becoming glued to it. All are heterodimers with two transmembrane polypeptides: the alpha and beta chains. Integrin-microfilament complexes are clustered in fibroblasts and other mesenchymal cells to form structures called focal adhesions. Physical properties of the ECM can change various cellular activities.

Water in the ground substance of connective tissue is referred to as interstitial fluid and has an ion composition similar to that of blood plasma. Interstitial fluid also contains plasma proteins of low molecular weight that pass through the thin walls of the smallest blood vessels, the capillaries.

Edema is the excessive accumulation of interstitial fluid in connective tissue. Capillaries in connective tissue also bring nutrients required by cells and carry away their metabolic waste products. Interstitial fluid is the solvent for these substances.

Two main forces act on the water in capillaries:

• The hydrostatic pressure of the blood caused by the pumping action of the heart, which forces water out across the capillary wall
• The colloid osmotic pressure produced by plasma proteins such as albumin, which draws water back into the capillaries

Colloid osmotic pressure exerted by the blood proteins pulls back water forced out by hydrostatic pressure. Excess fluid drains continuously into lymphatic capillaries that eventually return it to the blood. Lymphatic capillaries originate in connective tissue as delicate endothelial tubes.

Types of Connective Tissue

Different combinations and densities of the cells, fibers, and other ECM components produce graded variations in histological structure within connective tissue. Descriptive names or classifications used for the various types of connective tissue typically denote either a structural characteristic or a major component. Major types of connective tissue include adipose tissue, cartilage and bone. Connective tissue proper is divided into two categories "loose" and "dense".

Connective Tissue Proper

Connective tissue proper is broadly classified as “loose” or “dense,” terms that refer to the amount of collagen present. Loose connective tissue is common, forming a layer beneath the epithelial lining of many organs and filling the spaces between fibers of muscle and nerve. Also called areolar tissue, the loose connective tissue typically contains cells, fibers, and ground substance in roughly equal parts. The most numerous cells are fibroblasts, but the other types of connective tissue cells are also normally found, along with nerves and small blood vessels. Collagen fibers predominate, but elastic and reticular fibers are also present. With at least a moderate amount of ground substance, loose connective tissue has a delicate consistency; it is flexible and not very resistant to stress.

• Dense connective tissue has similar components as loose connective tissue, but with fewer cells, mostly fibroblasts, and a clear predominance of bundled type I collagen fibers over ground substance. The abundance of collagen here protects organs and strengthens them structurally
  • In dense irregular connective tissue, bundles of collagen fibers appear randomly interwoven, with no definite orientation. The tough three-dimensional collagen network provides resistance to stress from all directions. Examples of dense irregular connective tissue include the deep dermis layer of skin and capsules surrounding most organs. Dense irregular and loose connective tissues are often closely associated, with the two types grading into each other and making distinctions between them somewhat arbitrary
  • Dense regular connective tissue consists mostly of type I collagen bundles and fibroblasts aligned in parallel for great resistance to prolonged or repeated stresses from the same direction. The best examples of dense regular connective tissue are the very strong and flexible tendons, cords connecting muscles to bones; aponeuroses, which are sheetlike tendons; and ligaments, bands or sheets that hold together components of the skeletal system. Consisting almost entirely of densely packed parallel collagen fibers separated by very little ground substance and having very few blood vessels, these inextensible structures are white in the fresh state with elongated nuclei of fibrocytes Cytoplasm in these “tendinocytes” is difficult to distinguish in H&E-stained preparations because it is very sparse and has acidophilia like that of the collagen. In aponeuroses, the parallel bundles of collagen exist as multiple layers alternating at 90° angles to one another. Some ligaments, such as the elastic ligaments along the vertebral column, contain besides collagen many parallel bundles of elastic fibers. On their outer surface tendons and ligaments have a layer of dense irregular connective tissue that is continuous with the outermost layers of the adjacent muscles and bones. Collagen bundles vary in size in different tendons and ligaments, but all regular connective tissue structures are poorly vascularized. Ligaments and tendons will be discussed again with bone, joints, and muscle.

Overuse of tendon–muscle units can result in tendonitis, characterized by inflammation of the tendons and their attachments to muscle. Damaged collagen bundles of the area are eventually repaired by fibroblasts. . Swelling and pain produced by inflammation affects motion range and can be relieved with anti-inflammatory agents, namely cortisone.

Reticular Tissue

Reticular tissue is characterized by abundant fibers of type III collagen forming a delicate network that supports various types of cells. This collagen is also known as reticulin and is produced by modified fibroblasts often called reticular cells that remain associated with and partially cover the fibers. The loose disposition of glycosylated reticular fibers provides a framework with specialized microenvironments for cells in hemopoietic tissue and some lymphoid organs (bone marrow, lymph nodes, and spleen). The resulting cell-lined system creates a meshwork for the passage of leukocytes and lymph. Macrophages and dendritic cells are also dispersed within these reticular tissues to monitor cells formed there or passing through and to remove debris.

Mucoid Tissue

Mucoid (or mucous) connective tissue is the principal component of the fetal umbilical cord, where it is referred to as Wharton’s jelly. With abundant ground substance composed chiefly of hyaluronan, mucoid tissue is gelatinous, with sparse collagen fibers and scattered fibroblasts. Included among the fibroblastic cells are many mesenchymal stem cells, which are being studied for their potential in regenerative medicine. Mucoid connective tissue is similar to the tissue found in the vitreous chambers of eyes and pulp cavities of young teeth.