Connective Tissue

Components of Connective Tissue
Connective tissue it consist 3 types of components:

'1) Cells of connective tissue
 * Fibroblasts


 * Adipocytes


 * Macrophage and Mononuclear Phagocyte System


 * Mast Cells


 * Plasma Cells


 * Leucocytes

2)	Fibers of connective tissus


 * Collagen


 * Reticular fibers


 * Elastic fibers

3)Ground substances of connective tissue

Connective tissue is mostly consisted by ECM=Extracellular matrix and not from cells like other tissues. Extracellular matrix consists of :
 * protein fibers
 * collagen
 * reticular fibers
 * elastic fibers
 * ground substance
 * anionic macromolecules:
 * •	glycos-aminoglycans
 * •	proteo-glycans
 * multi adhesive glycoproteins:
 * •	laminin
 * •	fibronectin (That stabilizes the ECM by binding to the receptor proteins  integrins on the surface of cells)

Role:
-	structural -     biological= a reservoir of factors that controls the growth and the differentiation -	Medium in which nutrients and metabolic wastes are exchange between cells and their blood supply ( due to its hydrated nature)

Origin:
Mesenchyme (Mesenchymal cells) -	develops from the mesoderm (middle layer of the embryo)

-	migrate from their site of origin surrounding all developing organs

-	develops into other types of structure such as:


 * blood cells
 * endothelial cells
 * muscle cells

CELLS OF CONNECTIVE TISSUE
The connective tissue is consisted of many different cells with different origin and different function as:

Fibroblasts
-Fibroblasts originate from Mesenchymal cells ,are the most common cells in connective tissue, and spend all their life to connective tissue.

-Synthesize:


 * collagen


 * elastin


 * glycos-aminoglycans


 * proteo-glycans


 * multi-adhesive glycoproteins

-Have two stage of activity and they can be differentiated microscopically:


 * fibroblast = active cell


 * -	large cell
 * -	ovoid, large, pale-stain nuclei with fine chromatin,
 * -	prominent nucleolus
 * -	Basophilic, abundant, irregular cytoplasm,
 * -	rich in rough ER,
 * -	and well developed App. Golgi


 * fibrocyte = dormant cell


 * -	small cell
 * -	smaller and heterochromatic, darker , elongated nuclei,
 * -	more acidophilic cytoplasm with less RER
 * -Are targets of various growth factors that influence growth and differentiation!

Medical application:
The regenerative capacity of connective tissue is responsible for repairing destroyed tissues:

Scar is formed by connective tissue (CT) after traumatic or inflammation injury.

Healing of surgical incisions depends on reparative capacity of Connective T:


 * The fibrocyte reverts to the fibroblast state


 * The myofibroblast, a cell with features of both fibroblast and smooth muscle cell plays a basal role in “wound contraction”.


 * Myofibroblasts have most of the morphological characteristics of  fibroblasts but contain a big quantity of  Actin microfilaments and myosin responsible for the wound healing

Adipocytes
-They are connective tissue cells, special storage of neutral fats and the producers of heat. They are called fat cells.

Macrophages and mononuclear phagocyte system'''
-Originate from hematopoietic stem cell in bone marrow, circulate in the blood and after move into connective tissue where remain and execute their function -They have different morphology, different name and different function in base of their location in specialized tissue:
 * in connective tissue = Macrophages .
 * defense cellsand produce chemotactic factors, cytokines and several enzymes. In case of inflammation macrophages are stimulated and are arranged in clusters forming epithelioid cells (they look like epithelial cells). Change their morphological characteristics and metabolism becoming activated with increase capacity of phagocytosis.
 * *  antigen-presenting cells. They have class II MHC molecules in their surface. The CD4 Helper lymphocytes are activated by the presentation of antigen on Class II MCH molecule and proliferate by mitosis. The daughter T-cell lymphocytes activate the B cells that also proliferate by mitosis and after that differentiate into plasma cells. These plasma cells secrete specific antibodies for the antigen presented.


 * in blood=monocytes. They  are precursors of macrophages. Usually are increasing in viral infections.
 * in Liver=Kuppfer cells They are macrophages.
 * in nerve tissue and central nervous system=Microglia cell. They are Macrophages.
 * in Lymph nodes=Dendritic cells. They are Antigen presenting cells and are important in immune respond
 * in Skin=Langerhans cells.They are Antigen presenting cells.

Mast cells
Masts cells originate from Hematopoietic stem cell, the progenitor cell in Bone marrow. This progenitor circulates in blood, cross the wall of venules and capillaries and they penetrate the connective tissue where they proliferate and differentiate. They are not found in blood. They are large oval or round connective tissue cells. Their cytoplasm is filled with basophilic secretory granules containing paracrine compounds that promote local inflammatory response as:
 * histamine – acts in allergic reaction.
 * heparine –acts locally as anticoagulant
 * serine proteases- activate various mediators of inflammation
 * eosinophil and neutrophil chemotactic factors. In special conditions as in anaphylactic reactions Eosinophil chemotactic factor attracts eosinophils in phase of allergic reaction.
 * leucotrienes C4, D4, SRS-A=Slow Reacting Substance of Anaphylaxis

The granules display metachromasia=they can change color of some basic dyes (ex.toluidine blue) from blue to purple or red. This happens due to the high content of acidic radicals in their glycosaminoglycans and they are poorly preserved by common fixatives (usually mast cells are difficult to identify). The small, central, spherical nucleus is usually obscured by the cytoplasmic granules Specific locations of masts cells: -Perivascular masts cells are near small blood vessels in skin and mesenteries. -Mucosal mast cells in mucosa lining in digestive tract and in respiratory tract

Medical application
Anaphylactic shock is an immediate hypersensitive reaction. It occurs very quickly, within a few minutes, after penetration by an antigen of an individual previously sensitized. When the allergen (antigen) enters for the first time in the organism, plasma cell produce a specific IgE ( immunoglobulin or antibody contra the specific allergen) that is bound to the surface of a Mast cell. When a second exposure to the same antigen occurs, these are bind to IgE on mast cell which is activated leading to degranulation. Liberated histamine causes contraction of smooth muscle mainly in the bronchioles, and dilates and increases the permeability of capillaries. Due to the action of histamine are the symptoms of dyspnea and blushing that appear in this condition. The Eosinophil Chemotactic Factor of Anaphylaxis attracts blood Eosinophils. So in this condition in the full blood count characteristically appears eosinophilia (absolute number of eosinophils more than 800). Due to the fact that mast cells are abundant in skin, respiratory and digestive system the mainly symptoms in anaphylactic shock are concentrated initially at these areas.

Plasma Cell
Morphology
 * Are large ovoid cells
 * With basophilic cytoplasm due to rich RER
 * eccentrically placed, not central nucleus
 * Nucleus is spherical with an appearance of «clock-face»
 * Juxtanuclear Golgi apparatus + centriole ( pale region of the cell)
 * Normal there are a few plasma cell in most connective tissue.
 * Their life spasm is only 10-20 days.

Medical application

 * Immune respond to an antigen. Plasma cells are derived from B Lymphocytes and are responsible for synthesis of imunoglobulins. These are the antibodies that are produced after the penetration of a bacterium or (in general) an antigen the organism. Each antibody is specific for each antigen.
 * Malignant transformation of plasma cells leads to Monoclonal Plasma cells. These are cells that secrete a monoclonal immunoglobulin. When these monoclonal plasma cells represent more than 30% of the total bone marrow cells  produce the hematological disease named Multiple myeloma with characteristic bone lytic lesions.

Leucocytes
Originate from hematopoietic stem cell in the bone marrow, move to the connective tissue where they reside for a few days. They are transient cells of most connective tissue and usually die by apoptosis. Leukocytes migrate from the blood vessels by diapedesis. Diapedesis is increased during inflammation (defensive reaction against foreign substances or bacteria). Sings for inflammation are redness, swelling, heat and pain. Leucocytes do not return to the blood after arriving in connective tissue except lymphocytes.

Collagen
-It is a family of proteins!

-The most abundant protein in human body (30% of the body weight)

-We have more than 20 types of collagen in function of the degree of rigidity, elasticity and strength the cell type (which produce the collagen), the molecular composition, the morphological characteristics, function and distribution.

Classification
According to their structure + Function are classified in 4 categories:

1)	COLLAGEN that form FIBLILS (Visible by optical microscopy)


 * Type I	   -Skin, Tendon, Bone, dentin        -Resistance to tension


 * Type II       -Cartilage, vitreous body	         -Resistance to pressure


 * Type III      -Skin, muscle, blood vessels        -Structural maintenance in expansible organs


 * Type V        -Fetal tissues, Skin, bone          - Participate in type I  Collagen function


 * Type XI       - Cartilage	                	 - Participate in type II  Collagen function

The molecules of this collagen:


 * aggregate to form fibrils clearly visible in optical microscopy


 * collagen type I is the most abundant in organism


 * make the structures of the tissues named “ collagen fibers” forming tendons, organ capsules and dermis

2)	FIBRIL associated COLLAGENS ( Not visible detected by immunochemistry)


 * Type IX   - Cartilage, vitreous body      -Bound glycos-aminoglicans and is associated with Collagen type II


 * Type XII  - Embryonic tendon, Skin      -Interacts with type II collagen

These types of collagens:
 * Type XIV-Fetal skin, tendon


 * Are short structures


 * That binds the surface of collagen fibrils to one another and to other component of the ECM


 * Are also known as FACIT: Fibril Associated Collagens with Interrupted Triple helices

3)	COLLAGEN that forms ANCHORING FIBRILS ( not visible detected by immunochemistry)

This type of collagen:
 * Type VII -Epithelia	 -Anchors skin, epidermal basal lamina to underlying stroma


 * Bind the basal lamina to reticular fibers in the underlying connective tissue

4)	COLLAGEN that forms NETWORKS (not visible detected by immunochemistry)


 * Type IV – All basement membranes     - Support of delicate structures and - Filtration

This type of collagen *constitutes the major structural component of basal lamina

Synthesis
Collagen synthesis can be done in different cells as fibroblast, chondroblast, osteoblast and odontoblast.
 * In ribosome on RER is produced the procollagen “a chain”
 * This is intertwine (πλέκεται) to make triple helices and held together by hydrogen bonds and hydrophobic interactions
 * Every third amino-acid in “a chain” is glycine
 * Two other small amino-acids abundant in collagen are hydroxylated and form hydroxyproline and hydroxylysine.
 * The 3 “a chain” forms a rod-like procollagen molecule and can be:
 * -homotrimetric = when the 3 “a chain” are  identical
 * -heterotrimetric = when the 2 or all 3 “a chains” different

Collagen fibrils:
 * In collagen I, II and III the procollagen molecules are aggregate and become packed together forming FIBRILS
 * are thin, elongated structures with diameter =20-90 nm
 * have transverse striations with periodicity (64-68nm)
 * this is caused by the regular overlapping arrangement of the collagen molecules

-In some collagen types (V, XI) fibrils associate further with FACIT collagen to form FIBERS

-In collagen type I the fibers can form large BUNDLES

-In collagen type II (present in cartilage) the fibrils does NOT form fibers or bundles

-Collagen type IV (present in all membranes) assembles as a lattice-like network in the basal lamina

Microscopic examination: -In spite of «the fresh collagen fibers» are colorless strands, when appears in increased numbers for ex. In tendons, they appear white -In the light microscope collagen fibers are acidophilic and they stain:
 * Pink with Eosin
 * bleu with Malory trichrome stain
 * green with Masson trichrome stain
 * red with Sirius red

Medical application

 * Osteogenesis Imperfecta= A mutation in a single amino acid for example in glycine in colagen type I. Patients show spontaneous fractures and cardiac insufficiency
 * Progressive systemic sclerosis= over accumulation of collagen (fibrosis).
 * keloid= local swelling in the place of scars of the skin
 * Scurvy= Deficiency of Vit C is characterized by degeneration of connective tissue. In Vit C deficiency the fibroblasts synthetize defective collagen (In the hydroxylation of prolyne) Symptoms are loss of teeth and bleedings. Treatment by fresh food, particularly citrus fruit rich in vitamin C.

Reticular fibers
Reticular fibers are consisted mainly by collagen type III and forms thin and extensive network around the parenchymal cells of various organs for example liver and endocrine gland and in Hematopoietic organs for example spleen, lymph nodes, red bone marrow.

They are NOT visible in H&E heamtoxyline& Eosin preparation. They are stained BLACK by silver salts and are also called argyrophilic. They are also PAS positive.

Elastic Fibers
-Are thinner than the average collagen fiber

-Form sparse networks interspersed with collagen bundles in many organns as the wall of large arteries

- The major functional property is to give elasticity (resilient) to the organs

-Developing of E.F is made in 3 stages:

1)	First stage:


 * Fibrillin: a large glycoprotein formed from a core of 10-nm microfibrils


 * Fibrillin binds Elastin = forms scaffolding (σύστημα σκαλωσιάς) necessary for deposition of elastin

pulling forces
 * Microfibrils and fibrillin alone are used in some organs such as to hold in place the lens of the eye. Such Microfibrils are not elastic but are highly resistant to


 * Defective fibrillin = leads to th eproduction of fragmented elastic fibrils

2)	Second Stage:


 * deposition of elastin between microfibrils forming larger fibers


 * Elastin molecules:

-are globular and

-are secreted by fibroblasts in connective tissue and by smooth muscle cells in walls of blood vessels

-are rich in glycine and prolyne with many regions

-polymerize to form fibers or sheet-like structures

-contains 2 amino acids desmosine and isodesmosine which are produced when cross-links are formed among 4 lysine residues in different elastic molecules

-cross-links formed in lysine residues are catalyzed by lysil oxidase

-is resistant to digestion by most proteases

-is easily hydrolyzed by pancreatic elastase

3)	Third stage:

-Mature elastic fibers is produces by accumulation of elastin, which further surrounded by a thin sheath of microfilaments


 * Elastic lamellae= in the wall of large blood vessels, elastin occurs as a fenestrated sheet named elastic lamellae or elastic sheet


 * Microfibrils of fibrillin in some organs are used alone as in eye to hold in place the lens

'''How we color elastic fibers? ''' -Is difficult to be demonstrated by H&E

-Is demonstrated with Aldehyde Fuscin which stains elastin a dark magenta

Medical application
Syndrom Marfan: mutation in the fibrillin gene (protein that produce the scaffolding necessary for elastin)

The patient with Syndrome Marfan:

-Is tall and thin

-His tissues are not resistant ( lack of the resistance in tissue rich in elastic fibers)

-His big vessels like aorta usually have aneurysm that is very dangerous to be dissected

GROUND SUBSTANCES
The ground Substance of ECM is highly hydrated, transparent complex mixture of macromolecules:


 * 1) Glycos-aminoglycans (GAGs)


 * 2) Proteo-glycans


 * 3) Multi adhesive glycoproteins

-Fills the space between cells and fibers and

-Acts as lubricant and barrier to the penetration of invaders

Glycos-aminoglycans (GAGs)
-Originally called mucopolysacharides

-Formed from repeating disaccharide units composed from:

- Hexozamine   +      Urotic acid


 * glucosamin => glucuronic acid


 * galactozamine=>Iduronic acid

'''Hyaluronic acid is the most ubiquitous GAG '''

-With a molecular weight from 100-1000KDa

-Is a long polymer of a disaccharide glucosamine-glucuronate

-Is synthesized directly into ECM by the enzyme hyaluronate synthetase (located in the membrane of many cells)

-Forms a dense, viscous network of polymers

-This binds a big amount of water giving it a role:

1.In diffusion of molecules in connective tissue

2.In lubricating various organs and joints

-is found in umbilical cord, synovial fluid, vitreous humor, carilage

All other GAGs are much smaller 10-40kDa

-Are attached to proteins (Proteo-glycans)

-Are synthesized in Golgi complexes

-Are rich in sulfate

-are intense hydrophilic and viscous

- are polyanions and bind cations ( usually Sodium)

'''The four main GAGs which have different disaccharide unit and different tissue distribution are: '''

1.	Chondroitin sulfates ( 4 and 6) 

D Glucuronic acid + D Galactozamine  ->Cartilage, Bone, Cornea, Skin, Aorta

'''2.	Dermatan Sulfate '''

L Iduronic acid  or D Glucuronic acid + D Galactozamine   ->Skin, Tendon, Aorta

3.	Heparan Sulfate	

D Glucuronic acid or L Iduronic acid + D Galactozamine    -> Aorta, Lung, Liver,basal lamina 4.	Keratan Sulfate		

a) D Galactose + D Galactozamine  -> Cornea

b)D Galactose + D Glucosamine      -> Skeleton, nucleus pulposus, annulus fibrosus

Proteo-glycans
-Are composed of a core protein + GAG/s

-Are synthesized on RER

-Mature in the Golgi and

-Secreted from cells by exocytosis

The main differences between Proteoglicans and Glycoproteins are:


 * Proteoglycans:
 * -Contain a core protein as a vertical rod


 * -GAGs are covalently bound


 * -GASs are unbranched polysaccharide


 * -Contain a greater amount of carbohydrate (Than glycoproteins)


 * -Can be pictured as a “test tube brush”


 * Glycoproteins


 * -Are globular protein molecule


 * -Chains of monosaccharides are covalently attached


 * -Polypeptide content is greater than polysaccharide content

IN CARTILAGE:

-The core proteins of proteoglycans are bounded via small link proteins to hyaluronic acid producing proteoglycans aggregates -the acidic group of proteoglycans-> binds the basic amino acid residues of collagen

-Proteoglycans are distinguised for their diversity and are proteoglycans of :


 * Extracellular Matrix (ECM) . Best example is Aggrecan :


 * -is one of the most important ECM proteoglycans


 * -Is the dominant proteoglycan in cartilage


 * -The core protein has several chondroitin sulfate and keratin sulfate chains


 * - And is bound via a link protein to hyaluronic acid 


 * Cell Surface. Best example is Syndecan.


 * -Is a cell surface proteoglycan


 * -Is present on many types of cells, particularly epithelial cells


 * -The core protein spans the plasma membrane with a short cytoplasmic extension ->Heparan sulfate chains are attached to the extracellular extension

Multiadhesive glycoproteins
-Have attached carbohydrates that are usually branched	.

-The protein component predominates

-ROLE: in adhesion of cells to their substrate

1) Fibronectin


 * Is synthetized by fibroblasts and epithelial cells


 * Is a dimeric molecule with binding sites for


 * -Collagens


 * -GAGs


 * -Integrins of cell membranes


 * For this reason is named Multiadhesive glycoprotein


 * All these interactions lead to normal Cell Adhesion and migration


 * Fibronection is as a network in the intercellular spaces of many tissues

2) Laminin


 * is a trimetric cross-shaped glycoprotein with binding sites for


 * -Collagen type IV


 * -GADs


 * -Integrins


 * participate in adhesion of epithelium cells to basal lamina

3) Integrins = Matrix receptor


 * Are cell-surface molecule that bind to


 * -Collagen


 * -Fibronectin


 * -Laminin


 * Are transmembrane receptors  or linker proteins and


 * Interact also with cytoskeleton  Actin microfilaments in the presence of Talin and Vinculin


 * These receptor connect the components of ECM (collagen, Fibronectin, Laminin) with the intracellular components (actin via Talin)

Interstitial fluid

-In connective tissue there is a small quantity of free fluid

-Is similar to blood plasma

-Contains a small quantity of plasma proteins of low molecular that pass through the cappillay walls due to hydrostatic pressure of the blood

-Contains almost the 30% of  total plasma proteins of the body

Medical application
Edema: Is the accumulation of water in the extracellular spaces and is possible to be produced in:


 * Venous or lymphatic obstructions due to


 * -Parasitic plugs


 * -Tumor cells


 * -Chronic starvation


 * Decrease of venous blood flow due to congestive hard failure


 * Decrease in colloid osmotic pressure due to deficiency of plasma protein


 * Increased of permeability of blood capillary endothelium due to


 * -Chemical or mechanical injure


 * -Allergic reaction after production of histamine

TYPES OF CONNECTIVE TISSUE
1) LOOSE (ALVEOLAR)


 * Is a very common type


 * Has a delicate consistency and is


 * -Flexible


 * -Well vascularized


 * -And not very resistant to stress


 * Support many structures that are normally under pressure


 * Supports epithelial tissue


 * -In glands


 * -In the mucous membranes


 * -In peritoneal and pleural cavities


 * Forms a -layer around small blood and lymphatic vessels and papillary layer of dermis in hypodermis
 * Fills the spaces between muscle and fibers
 * Is also called areolar tissue (has all components of connective tissue in equal parts

 2) DENSE


 * Offer resistant and protection


 * Has the same components with the loose connective tissue but Fewer cells and more collagen fibers


 * Is less flexible


 * Is more resistant to stress


 * Is of two different type in function of orientation of collagen fibers:


 * a. Irregular

-Fibers in bundles without orientation -form a 3-dimentional network -provide resistance to stress from all directions -is found close associated with loose connective tissue


 * b. Regular

-fibers in bundles are arranged with linear orientation -in the same direction with the exerted stress -and offers great resistance to traction forces -is found in tendons and ligaments -due to the fact that are rich in collagen fibers are white and inextensible -they have parallel closely packed bundles of collagen and -a very small quantity of ground substance -a few fibrocytes with elongated nuclei and sparse cytoplasm that is not revealed in H&E because it stains the same color with fibers -The collagen bundles of tendons are enveloped by small amounts of loose connective tissue, with small vessels and nerves. -are poorly vascularized so the repair of damage is very slow -externally the tendon is surrounded by a sheath of dense irregular tissue -Synovial cells of Mesenchymal origin form a sheath, from 2 layers. -The space between these two layers is filled with fluid similar to the fluid of synovial joints rich in water, proteins,Hyaluronate and other GAGs -Acts as lubricant

3) SPECILIZED

Is of two types

a. Reticular connective tissue
 * Is a 3-dimesnional network


 * formed by reticular fibers of type III collagen


 * produced by reticular cells ( specialized fibroblasts)


 * heavily glycosylated reticular fibers provide the architectural framework for cell attachment


 * is found in hematopoietic and lymphoid organs : bone marrow, Lymph nods, spleen


 * the reticular cells are dispersed along this framework forming a spongelike structure in which cells and fluids are freely mobile

b. Mucous connective tissue
 * is found in the umbilical cord and fetal tissue


 * has an abundant ground substance


 * composed mostly of hyaluronic acid

'''
 * making it a jellylike tissue named '''Wharton’s Jelly (in umbilical cord)
 * is found also in pulp cavity of young teeth