CONNECTIVE TISSUES

CONTENT

INTRODUCTION CLASSIFICATION
- Connective tissue proper

CELLS IN CONNECTIVE TISSUES

- Fibroblasts -Macrophages -Adipose cells - Mast ells

TYPES OF FIBERS
- Collagen fibers - Reticular fibers - Elastic fibers

GROUND SUBSTANCE
- Proteoglycans - Glyoproteins

INTRODUCTION
These are group of tissues predominantly composed of intercellular material (matrix). They are derived from the mesoderm. Connective tissues play many important roles in the body both structural and defensive. Early during embryological development the ectoderm and endoderm become separated by the third germ layer the Mesoderm. The tissues formed by this layer is called Mesenchyme mesos means middle and enchyma means infusion. Connective tissue consists of cells and a extra cellular matrix that includes extracellular fibers, substance and tissue fluids.

CLASSIFICATION OF CONNECTIVE TISSUES

Classification depends upon composition organ ization of the cellular and extra cellular components special functions

CONNECTIVE TISSUE PROPER Loose connective tissue Dense connective tissue -Irregular -Regular

SPECIALIZED CONNECTIVE TISSUE Adipose tissue Blood Bone Cartilage Hemopoietic tissue Lymphatic tissue

EMBRYONIC CONNECTIVE TISSUE Mesenchyme Mucous connective tissue

Connective Tissues Proper has two sub types: Loose connective tissue Dense connective tissue

Loose Connective Tissue or Areolar Tissue

These cells are characterized by loosely arranged fibers, abundance of cells, thin and relatively sparse, viscous gel like consistency..

Dense Connective Tissue

On bases of arrangement of fibers it is further sub divided as: Dense Irregu lar Connective Tissue Dense Regular Connective Tissue

Dense Irregular Connective Tissue

These are found in the region which experience mechanical stress and where protection is to be provided. Contain high proportion of collagen which forms thick bundles oriented in yarious directions. Due to high proportion of collagenous fibers it provides high strength. Cell population is less and is typically of a single type fibroblast. Active fibroblast are few. Mostly are flattened with heterochromatic nuclei. Found in dermis, connective tissue sheaths of muscle, nerve and large blood vessels.

Dense Regular Connective Tissue These are characteri zed by ordered and densely packed array of fibers and cells. These are the main functional component of tendons, ligaments and aponeuroses.

Tendons They are cord like structure that join muscle to bone. It consists of parallel bundles of collagenous fiber between which are row of fibroblast present. The substance of tendon is surrounded by a thin connective ti ssue capsule epitendineum.

Ligament These are similar to tendon. They consist of fibers and fibrob last arranged parallel direction. The fibers are less regularly arranged as compared to that of tendon function of ligament is to join bone to bone.

Aponeuroses They are much more like flattened tendon. The fibers are arranged in multiple layers. These are sheet of dense connective tissue. It bears tensile forces directly or indirectly from skeletal muscle to other muscle cartilage or bone. The bundles of collagen fibers in a layer are arranged at 90 degree to neighboring layers.

CELLS PRESENT IN CONNECTIVE TISSUES

Connective tissue cells can be classified as: Fixed Wandering

Fixed These cells are relatively stable permanent residents of the tissue. These cells are: Fibroblasts Macrophages Adipose cells Mast cells

Wandering They migrate into tissue from blood on response to specific stimuli. These cells are: Lymphocytes Monocytes Neutrophils Eosinophils Basophils Plasma cells

FIBROBLAST These are the principal cells of connective tissue. They are responsible for the synthesis of collagen, elastin and reticular fibers and the complex carbohydrates of the ground substance. Its structure is flattened, irregular in outline having branching processes.

Profile is spindle shaped. Active fibroblast has an oval, pale-staining nucleus and has more cytoplasm. Nucleus of active fibroblast is euchromatic (opened - faced). Old fibroblasts are elongated with less cytoplasm. Nucleus of old, inactive fibroblast is flattened, heterochromatic (closed - faced). Fibroblasts are separated from one another by extracellular matrix components. Exception is embryonic tissue and periodontal ligament were there is cell to cell contact. Originate from mesenchymal cells. Once they are differentiated they are replicate by mitosis. Cultured fibroblast undergoes 50-55 division. Fibroblast from long Iived species divides more then fibroblast from short lived species. Gradual loss of telomere DNA is the onset of senescence. Accumulation of oxidative damage to DNA & protein contribute to senescence. Cultured fibroblasts that become senescent never die.

MACROPHAGES A.lso known as Histiocytes or Clasmatocytes These ells are relative ly larger in size 15 - 20 11m diameter. Nuclei are usually indented or kidney shaped. Cytoplasm is mildly basophilic and typically has a "frothy" appearance under light microscope. Surface of macrophages have numerous folds and fingers like projection. These folds and fingers helps in Phagocytosis

ADIPOSE CELLS When occurring singly the cells are oval Qr spherical in shape, but when mutually compressed they become Polygonal in shape. Each cell consists of peripheral rim of cytoplasm, in which the nucleus is embedded surrounding a single large central globule of- fat. They are fixed and stained by osmium tetroxide and specially coloured by alcoholic solution of certain dyes - sudan III, sudan black. Adipose cells when acumulated in large number are called Adipose tissue.

MAST CELLS Also known as Mastocytes or Histaminocytes. These cells are important defensive cells. Their shapes are round or oval about 12 micro meter in diameter. It has many Filopodia extending from cell surface. It's cytoplasm is filled with large granules. It has a centrally placed nucleus. It is stained by Periodic Acid Schiff PAS) method. Mast cells are distributed in vicinity of small blood vessels.

TYPES OF FIBERS Each fiber type is produced by the fibroblast. They are composed of protein formed by long peptide chains Fibers are mainly of three types: Collagen fibers Reticular fibers Elastic fibers

Collagen Fibers It forms the most abundant fibers of connective tissue. They are very flexible, have high tensile strength. Provide strength and support to the tissue. Appear as wavy structures of variabIe width and indeterminate length. They stain readily with eosin and other acid dyes. Linder electron microscope they appear as bundle or bundles of fine thread like subunit called collagen fibril. Collagen fibrils within a fiber are relatively of uniform in length. Developing or immature tissue fibrils are as small as 15-20 nm in diameter fibrils of dense regular connective tissue like tendon measure up to 200nm in diameter. They constitute the most abundant protein found in the body. Collagen family consists of 30 different genes. This produces 19 known collagens. They are composed of 3 polypeptide alpha chains coiled around each other. This forms a typical collagen triple - heIix configuration. They include the presence of amino. acid glycin in every third position. Collagen molecules are called tropocollagen. They are 300nm long and 1.5nm in thickness. They exhibit a sequence of closely spaced transverse band. These repeat after every 68nm along the length of the fibril.

Variation among collagens includes :Difference in the assembly of the basic polypeptide chains. Different length of triple helix. Interruption in the helix and difference in the terminations of the helical domains. Sylthesis of collagen involves both event that occur Within and outside the fibroblast. Within the fibroblast precursor of collagen is formed called procollagen. The actual collagen is formed outside the fibroblast. Involving enzymatic activity at the plasma membrane. Collagen molecules formed are aliened to form fibril under the guidance of ceII.

INTRACELLULAR EVENTS First cleavage of polypeptide chain occurs. Then hydroxylation of proline and lysine takes place. Addition of O-linked sugar group to some ofthe hydroxylysine residues and N-linked sugar to the terminal ends take place. Formation of triple helix by 3 polypeptide chains except at the terminals occurs. Then Formation of interchain and intrachain hydrogen bonds that influence the shape of the molecule. Now the resultant molecule is called procollagen. Procollagen moves out of cell by exocytosis of secretory vesicles.

EXTRACELLULAR EVENTS Firstly procallagen is converted to collagen by procollagen pentidase. Then the secreted molecules concentrate at an indentation on the cell surface. Then assemble in row occurs to cross link.

TYPE OF COLLAGEN
TYPES I COMPOSITION -1, -2 LOCATION Skin,bone,dentin, cementum, tendon,ligament II -1 Cartilage, intervertebral disk III -1 Embryonic connective tissue, pulp, skin,bloodvessels IV -1, -2, -3, -4, -5, -6 Basement membrane Structural network of basement membrane V -1, -2, -3 Basement membrane,blood vessel, ligament, dentin,periodontal tissue VI -1, -2, -3 Ligament, skin, cartilage VII -1 Epithelium (skin,mucosa) Bridging between cells and matrix Strengthens epithelialconnective tissue junction VIII IX -1, -2 -1, -2, -3 Cornea Cartilage, vitreous humor Tissue support Attaches functional group to surface of type II fibril Provides tensile strength FUNCTION Resistance to force, tension, stress Tensile strength to connective tissue Tensile strength to connective tissue

-1

Hypertrophic zone of cartilage growth plate

Calcium binding

XI

-1, -2

Cartilage, vitreous humor

Provides tensile strength Modulates fibril interactions Cell-matrix, cellcell adhesion

XII

-1

Widespread in connective tissue

XIII

-1

Cell surface, focal adhesions, intercalated disks

XIV

-1

Widespread in connective tissue

Modulate fibril interaction Proteolytic release of antiangiogenic factor unknown

XV

-1

Endothelial basement membrane

XVI

-1

Endothelial muscle,basement membrane

XVII

-1

hemidesmosomes

Cell attachment to matrix

XVIII

-1

Endothelial basement membrane

unknown

XIX

-1

Endothelial muscle,basement membrane

unknown

INHERITED DISEASES INVOLVING COLLAGENS I. Osteogenisis imperfecta or brittle bone diseases due to deficiency of Type I 2. Ehlers Danlos syndrome (hyper extensible skin, hyper mobile joint and tissue fragility) due to defi ciency of Type I, III,Y 3. Stickler syndrome retinal detachment, cataract, hearing loss, joint problem, cleft palate, facial and dental abnormalities) due to deficiency of Type II, XI 4. Epidermolysis bullosa (separation of epidermis and dermis) due to deficiency of Type VII, XVII

RETICULAR FIBERS It provides supporting framework for cellular constituents of various tissues and organs of the body. They are arranged in mesh like pattern or network. They are closely related to collagen fibers as it also contains collagen fibrils. They are composed of Type III c0l1agen. Type IV may be also associated. They do not bundle to form thick fibers. Found at the boundary of loose connective tissue. They are also present as a supporting stroma in hemopoietic and lymphatic tissues. These type of reticular fibers are produced by special cells call ed reticular cells. Rest all reticular fibers are produced by fibroblast.

ELASTIC FIBERS Elastic fibers provide tissues with the ability to respond to stretch and distension. They are typically thinner then collagen fibers. They are arranged in branching pattern forming a three dimensional network. Fibers are interwoven with collagen fibers this prevent tearing from excessive stretching. They are produced by fibroblast and smooth muscle cells. Elastin It's a protein that is rich in proline and glycine but poor in hydroxyproline and completely lacks hydroxylysine. Microfibril are a fibular glycoprotein that is relatively straight and thin measuring 12 nm in diameter. They are found in ligament vertebral column and neck and vocal cord.

GROUND SUBSTANCE OR INTERFIBULLAR SUBSTANCE It is the component that occupies the space between the cells and fibers. It is a colourless transparent substance. It is viscous clear substance having a slippery feel. it has high water content. It provides mechanism for regulating tissue water content & diffusion of nutrition, waste and other molecules. Physical properties of ground substance are same whether viscous nature in connective tissue or turgid character in cartilage. It is stained by periodic acid schiff (PAS) method. In routine H&E preparation ground substance is always lost due to extraction during fixation and dehydration. Functions as a molecular sieve through which nutrients diffuse between blood capillaries and cells.

Glycosaminoglycan is a major component.

GLYCOSAMINOGLYCANS (GAG) these are unbranched chain of repeating disaccharide units. Each unit carry one or more negatively charged group. This negative charge helps in osmotic activity. Due to high water attraction it forms hydrated gel. This causes swelling pressure and helps in keeping fibriIs apart and causes stiffness. On the basis of Specific sugar residues, Nature of linkages and Degree of sulfation Glycosaminoglycans are of seven distinct types. 1. Hyaluronic acid 2.Chondroitin sulfate 3. Dermatan sulfate 5. Heparin 6. Keratan sulphate

HYALURONIC ACID These are found in abundance in embryonic tissues and cartilage also found in all ECM. It has simple structure of extremely long chains of non-sulfated disaccharides. It forms. The keystone in aggregation of proteoglycans and link protein. Found in abundance in embryonic tissues and catilage also found in all extra cellular matrix. It has a simple struture of extremely long chains of non-sulfated disaccharides. It forms the keystone in aggregation of proteoglycans and link protein.

Hyaluronic acid is not bound to core protein and other glycosaminoglycans are bound to the core protein forming a bottle like structure.

CHONDROITIN SULPHATES 4 & 6 (CS4, CS6) They are labeled as according to the predominant component sulphate (4 & 6). Found in cartilage in very high concentration and in most extra cellular matrix. Most are mixed or hybrid AGAGs. Undersulphated chondroitinsulphates is CS4 found in cornea. While properly sulphated chondroitin sulphates is CS6 are found in vitreous body and oversulphated chondroitin sulphates is CSO which are found in cartilage of fishes.

KERATAN SULPHATES (KS) Their polymer back bone is identical to that of chondroitin. They are Present in age cartilage and intervertibral discs and cornea.

DERMA SULPHATES (DS) They are formed by CS4. OS aggregates with itself in solution, this causes the organization of most extra cellular matrix. They are found in very high concentration in tissue.

HEPARAN SULPHATES It is a important constituent of basement membrane. They interact directly with constituents of extra cellular matrix. They are usually located on skin fibroblast.

HEPARIN They are made-up of repeating disaccharide units contains glucosamine and two uronic acids. They are usually located in mast cells, liver lung and skin.

GLYCOPROTEIN There may be one or more carbohydrate chains covalently linked to a protein. The chains may be neutral or negatively charged. They are frequently branched. They have a acihesivc properties. One of their primary function is to bind cells to extracellular matrix elements.

SOME FUNCTIONS OF GLYCOPROTEIN AND PROTEOGLYCANS They act as structural component of the extra cellular matrix. They have specific infraction with collagen, elastin, fibronectin and laminin. They facilitates cell migration. Thay play a role in making cartilage stress bearing. They acts as anticoagu lant. They are components of plasma membrane where they may act as receptors and participate in cell adhesion.

FILAMENTOUS PROTEIN WITH ADHESIVE PROPERTIES These are structural proteins, which contains molecules that mediate adhesion between cells and extra cellular matrix. These are glycoproteins, which helps cell to adhere at the appropriate matrix structure. They contain signaling molecules which are detected by the cell surface receptors. They initiates changes within the cytoplasm. The best known filamentous proteins are Fibronectin, Laminin and Tenascin.

FINRONECTIN Larce glycoprotein molecule consisting of a dimer held together by disulphide link. Each subunit is composed of a string of large repetitive domain joined by flexible region. These bears binding sites for collagen and cell surface receptors. They are bound in onkrly fashion to cell su rface forming fibronectin filaments.

FIBRONEXUS It is the term use to describe the morphologic relationship between Intracellular filament, Cell membrane, Extracellular filament and to the sticky attachment glycoprotein fibronectin. The binding of myofibroblast to each other causes fibronexus to transmit the collective forces generated by the contraction of actin microfilaments throughout the granulation tissue. This causes wound

contraction. Fibronexus are associated with fibroblast in transseptal fiber region of gingiva. New studies show fibronexus are present only in inflamed gingiva.

LAMININ Large flexible cruciform molecule composed of three polypeptide chains (a, 13, y). Their terminal two third are wound round each other to form the stem of cross. The free end forms the upright members and transverse members. It bears binding sites for a number of extra cellular matrix molecules like heparan sulphate, Type IV collagen, and laminin.

TENASCIN Large glycoprotein consisting of six subunits joined at one end to form a radiating structure which resembles spoke of a wheel. It is abundant in embryonic tissues. These are fo und less in adult tissues. They play important role in guiding cell migration. Tenascin is a glycoprotein associated with the extra-cellular matrix and the surface of some cell types. During early stages of neural crest migration, tenascin is observed in a dense matrix surrounding premigratory cranial neural crest cells. During advanced neural crP-:t migration, tenasc in immunoreactivity colocaUzed with and appeared to be on the surface of migrating neural crest cells. At later stages; tenascin is present around the otic vesicles, retina, lens, and in an interstitial matrix in the region ofthe branchial arches. At the level of the occipital somites, tenascin immunoreactivity is present around the neural tube, notochord and on the basal surface of the ectoderm. Tenascin is requ ired for proper cranial neural crest migration.

PROTEOGLYCANS These are very large macromolecules of protein to which many glyscosaminoglycans molecules are covalently bounded. Proteoglycan binds growth factors and cytokines. They are essential co-receptor for growth factor and are involved in transmembrane signaling. Five proteoglycan are of particular interest these are Decorin, Versican, Perlecan, Syndecan and CD44.

DECORIN They are called so because it binds to collagen and is visualized as "decoration" on collagtn fibril. They play an important role in regu lation of growth, diameter or both of collagn fibril.

VERSICAN They are large proteoglycan that assist in bonding cell surface glycoprotein to extra cellular matrix.

PERLECAN They are cell surface proteoglycan. Its name arises because the large core protein containing number of globular domains which form pearl necklace like structure. It binds fibroncctin that helps in anchoring fibroblast to extra cellular matrix.

SYNDECAN It has an extracellular domain, membrane-spanning domain and cytoplasmic domain. It binel collagen and other extracellular glycoproteins.

CD44 It has transmembrane structure which is capable of binding to fibronectin, laminin and collgen.

CLASSIFICATION OF PROTEOGYCANS According to the shape and size of the protein core Proteoglycans are classified into 3 groups: I. Small 2. Large 3. Very large

SMALL PROTEOGLYCANS They are attached to the family of globular protein of very small size amino acid and gene structure. Electron microscopically they appears as Tadpole - shaped with globular protein at the head and one or two glycan chains at the tail.

LARGE PROTEOGLYCANS Their molecule contains one or two globular protein domains. They are connected via a polypeptide chain. To which 5 - 10 acidglycosaminoglycans are attached.

VERY LARGE PROTEOGLYCANS These are complex molecules with up to three globular regions linked by a polypeptide chain. To which about 100 chondroitin sulphates and keratan sulphates chains are attached.

PROTEOGLYCANS COLLAGEN INTERACTIONS The proteogylcans interact with collagen rich fibril s. They sometimes completely encircle them. Very frequently they form bridge between neighboring fibrils. In cornea as many as 4 tibil s are bridged by the same filament of acidglycosaminoglycans. They acts as cross linking structure between proteoglycan cores which are attached to the collagen fibril.

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