Inflammation Doc Main

K.L.E SOCIETY`S INSTITUTE OF DENTAL SCIENCES, BENGALURU

DEPARTMENT OF PERIODONTICS

SEMINAR TOPIC

INFLAMMATION & ITS CHEMICAL MEDIATORS

PERIODONTAL WOUND HEALING & REPAIR

Date of presentation Date of submission

Presented by,

Dr.Aslam Abdul RahmanPG StudentDept of Periodontics

CONTENTS

Introduction & Definition

Types of inflammation

Signs of inflammation

Acute Inflammation

Chemical mediators of inflammation

Chronic Inflammation

Gingival inflammation

Periodontal inflammation

Conclusion

References

DEFINITION

Inflammation is defined as the local response of living mammalian tissues to injury

due to any agent.

INTRODUCTION

The same exogenous and endogenous stimuli that cause cell injury also elicit a

complex reaction in vascularised connective tissue called inflammation. Reduced to its

simplest terms, inflammation is a protective response intended to eliminate the initial cause of

cell injury as well as the necrotic cells and tissues resulting from the original insult.

The inflammatory response has many players. These include circulating cells and

plasma proteins, vascular wall cells and extra cellular matrix and its cells in the surrounding

connective tissue.

While the inflammatory response involves a complex set of highly orchestrated

events, the broad outlines are as follows: An initial inflammatory stimulus triggers the release

of chemical mediators from plasma or connective tissue cells. Such soluble mediators, acting

together or in sequence, amplify the initial inflammatory response and influence its evolution

by regulating the subsequent vascular and cellular responses. The inflammatory response is

terminated when the injurious stimulus is removed and the inflammatory mediators have been

dissipated, catabolised or inhibited.

TYPES OF INFLAMMATION

Inflammation is divided into acute and chronic patterns.

Acute inflammation is rapid in onset (seconds or minutes) and is of relatively short duration,

lasting for minutes, several hours, or a few days; its main characteristics are the exudation of

fluid and plasma proteins (edema) and the emigration of leukocytes, predominantly

neutrophils.

Chronic inflammation is of longer duration and is associated histologically with the presence

of lymphocytes and macrophages, the proliferation of blood vessels, fibrosis, and tissue

necrosis. Many factors modify the course and morphologic appearance of both acute

and chronic inflammation.

Components of inflammatory response

SIGNS OF INFLAMMATION

5 cardinal signs

Rubor (Redness)

Tumor (Swelling) CELSUS

Calor (Heat)

Dolor (Pain)

Functio laesa (Loss of function) VIRCHOW

ACUTE INFLAMMATION

Acute inflammation is the immediate and early response to injury designed to deliver

leukocytes to sites of injury. Once there, leukocytes clear any invading microbes and begin

the process of breaking down necrotic tissues.

Acute inflammation has three major components: (1) alterations in vascular caliber that lead

to an increase in blood flow; (2) structural changes in the microvasculature that permit

plasma proteins and leukocytes to leave the circulation; and (3) emigration of the

leukocytes from the microcirculation, their accumulation in the focus of injury, and their

activation to eliminate the offending agent.

Acute inflammatory reactions are triggered by a variety of stimuli:

Infections (bacterial, viral, parasitic) and microbial toxins

Trauma (blunt and penetrating)

Physical and chemical agents (thermal injury, e.g., burns or frostbite; irradiation;

some environmental chemicals)

Tissue necrosis (from any cause)

Foreign bodies (splinters, dirt, sutures)

Immune reactions (also called hypersensitivity reactions)

VASCULAR CHANGES (Lampugnani MG et al 1997)

Changes in Vascular Flow and Caliber

Changes in vascular flow and caliber begin early after injury and develop at varying rates

depending on the severity of the injury. The changes occur in the following order:

■ Vasodilation is one of the earliest manifestations of acute inflammation; sometimes, it

follows a transient constriction of arterioles, lasting a few seconds. Vasodilation first involves

the arterioles and then results in opening of new capillary beds in the area. Thus comes about

increased blood flow, which is the cause of the heat and the redness . Vasodilation is induced

by the action of several mediators, notably histamine and nitric oxide, on vascular smooth

Muscle.

■ Vasodilation is quickly followed by increased permeability,of the microvasculature, with

the outpouring of protein rich fluid into the extravascular tissue.

■ The loss of fluid results in concentration of red cells in small vessels and increased

viscosity of the blood, reflected by the presence of dilated small vessels packed with red cells

and slower blood flow, a condition termed stasis. With mild stimuli, stasis may not become

apparent until 15 to 30 minutes have elapsed, whereas with severe injury, stasis may occur in

a few minutes.

■ As stasis develops, leukocytes, principally neutrophils, accumulate along the vascular

endothelium. Leukocytes then stick to the endothelium, and soon afterward they migrate

through the vascular wall into the interstitial tissue.

Increased Vascular Permeability

(Vascular Leakage)

A hallmark of acute inflammation is increased vascular permeability leading to the escape of

a protein-rich fluid (exudate) into the extravascular tissue. The loss of protein from the

plasma reduces the intravascular osmotic pressure and increases the osmotic pressure of the

interstitial fluid.

Together with the increased hydrostatic pressure owing to increased blood flow through the

dilated vessels, this leads to a marked outflow of fluid and its accumulation in the interstitial

tissue . The net increase of extravascular fluid results in edema.

Normal fluid exchange and microvascular permeability are critically dependent on an intact

endothelium.

It can occur by :-

Formation of endothelial gaps in venules. This is the most common mechanism of

vascular leakage and is elicited by histamine, bradykinin, leukotrienes, the

neuropeptide substance P, and many other classes of chemical mediators. It occurs

rapidly after exposure to the mediator and is usually reversible and short-lived (15 to

30 minutes); it is thus known as the immediate transient response. Classically, this

type of leakage affects venules 20 to 601nn in diameter, leaving capillaries and

arterioles unaffected.

Direct endothelial injury, resulting in endothelial cell necrosis and detachment." This

effect is usually encountered in necrotizing injuries and is due to direct damage to the

endothelium by the injurious stimulus, as, for example, in severe burns or lytic

bacterial infections. Neutrophils that adhere to the endothelium (discussed below)

may also injure the endothelial cells. In most instances, leakage starts immediately

after injury and is sustained at a high level for several hours until the damaged vessels

are thrombosed or repaired. The reaction is known as the immediate sustained

response. A ll levels of the microcirculation are affected, including venules,

capillaries, and

arterioles. Endothelial cell detachment is often associated with platelet adhesion and

thrombosis.

Delayed prolonged leakage. This is a curious but relatively common type of

increased permeability that begins after a delay of 2 to 12 hours, lasts for several

hours or even days, and involves venules as well as capillaries. Such leakage is

caused, for example, by mild to moderate thermal injury, xradiation or ultraviolet

radiation, and certain bacterial toxins. Late-appearing sunburn is a good example of a

delayed reaction. The mechanism of such leakage is unclear. It may result from the

direct effect of the injurious agent, leading to delayed endothelial cell damage

(perhaps by apoptosis), or the effect of cytokines causing endothelial retraction.

Leukocyte-mediated endothelial injury. Leukocytes adhere to endothelium relatively

early in inflammation. Such leukocytes may be activated in the process, releasing

toxic oxygen species and proteolytic enzymes, which then cause endothelial injury or

detachment, resulting in increased permeability. In acute inflammation, this form of

injury is largely restricted to vascular sites, such as venules and pulmonary and

glomerular capillaries, where leukocytes adhere for prolonged periods to the

endothelium.'

Increased transcytosis across the endothelial cytoplasm. Transcytosis occurs across

channels consisting of clusters of interconnected, uncoated vesicles and vacuoles

called the vesiculovacuolar organelle, many of which are located close to

intercellular junctions. Certain factors, for example, vascular endothelial growth

factor (VEGF) , appear to cause vascular leakage by increasing the number and

perhaps the size of these channels." It has been claimed that this is also a mechanism

of increased permeability induced by histamine and most chemical mediators.

Leakage from new blood vessels, during repair, endothelial cells proliferate and form

new blood vessels, a process called angiogenesis. New vesselsprouts remain leaky

until the endothelial cells mature and form intercellular junctions. In addition, certain

factors that cause angiogenesis (e.g., VEGF) also increase vascular permeability,' and

endothelial cells in foci of angiogenesis have increased density of receptors for

vasoactive mediators, including histamine, substance P, and VEGF.'' All these factors

account for the edema that is characteristic of the early phases of healing that follow

inflammation

CELLULAR EVENTS: LEUKOCYTE EXTRAVASATION AND PHAGOCYTOSIS

The sequence of events in the extravasations of leukocytes from the vascular lumen to the

extra vascular space is divided into

Margination and rolling

Adhesion and transmigration between endothelial cells

Migration in interstitial tissues toward a chemotactic stimulus

Leukocyte Adhesion and Transmigration

Leukocyte adhesion and transmigration are regulated largely by the binding of

complementary adhesion molecules on the leukocyte and endothelial surfaces, and chemical

mediators chemoattractants and certain cytokines—affect these processes by modulating the

surface expression or avidity of such adhesion molecules. '' The adhesion receptors involved

belong to four molecular families—the selectins, the immunoglobulin superfamily, the

integrins, and mucin-like glycoproteins.

■ Selectins, so called because they are characterized by an extracellular N-terminal domain

related to sugar-binding mammalian lectins, consist of E-selectin (CD62E, previously known

as ELAM-1), which is confined to endothelium; P-selectin (CD62P, previously called

GMP140 or PADGEM), which is present in endothelium and platelets; and L-selectin

(CD62L, previously known by many names, including LAM-1), which is expressed on most

leukocyte types . Selectins bind, through their lectin domain, to sialylated forms of

oligosaccharides (e.g., sialylated Lewis X), which themselves are covalently bound to various

mucin-like glycoproteins (GIyCAM-1, PSGL-1, ESL-1, and CD34).

■ The immunoglobulin family molecules include two endothelial adhesion molecules: ICAM-

1 (intercellular

adhesion molecule 1) and VCAM-1 (vascular cell adhesion molecule 1). Both these

molecules serve as ligands for integrins found on leukocytes.

■ Integrins are transmembrane heterodimeric glycoproteins, made up of a and B chains, that

are expressed on many cell types and bind to ligands on endothelial cells, other leukocytes,

and the extracellular matrix. 20

The 13, integrins LFA-1 and Mac-1 (CD11a/CD18 and CD11b/CD18) bind to ICAM-1, and

the /31 integrins (such as VLA-4) bind VCAM-1.

■ Mucin-like glycoproteins, such as heparan sulfate, serve as ligands for the leukocyte

adhesion molecule called CD44. These glycoproteins are found in the extracellular matrix

and on cell surfaces.

The recruitment of leukocytes to sites of injury and infection is a multistep process involving

attachment of circulating leukocytes to endothelial cells and their migration through the

endothelium . The first events are the induction of adhesion molecules on endothelial cells, by

a number of mechanisms. Mediators such as histamine, thrombin, and platelet activating

factor (PAF) stimulate the redistribution of P-selectin from its normal intracellular stores in

granules (Weibel-Palade bodies) to the cell surface. Resident tissue macrophages, mast cells,

and endothelial cells respond to injurious agents by secreting the cytokines TNF, IL-1, and

chemokines (chemoattractant cytokines).

TNF and IL-1 act on the endothelial cells of postcapillary venules adjacent to the infection

and induce the expression of several adhesion molecules. Within 1 to 2 hours, the endothelial

cells begin to express E-selectin. Leukocytes express at the tips of their microvilli

carbohydrate ligands for the selectins, which bind to the endothelial selectins. These are low-

affinity interactions with a fast off-rate, and they are easily disrupted by the flowing blood.

As a result, the bound leukocytes detach and bind again, and thus begin to roll along the

endothelial surface.

TNF and IL-1 also induce endothelial expression of ligands for integrins, mainly VCAM-1

(the ligand for the VLA-4 integrin) and ICAM-1 (the ligand for the LFA-1 and Mac-1

integrins). Leukocytes normally express these integrins in alow-affinity state. Meanwhile,

chemokines that were produced at the site of injury enter the blood vessel, bind to endothelial

cell heparan sulfate glycosaminoglycans (labeled "proteoglycan", and are displayed at high

concentrations on the endothelial surface."

These chemokines act on the rolling leukocytes and activate the leukocytes. One of the

consequences of activation is the conversion of VLA-4 and LFA-1 integrins on the

leukocytes to a high-affinity state. The

combination of induced expression of integrin ligands on the endothelium and activation of

integrins on the leukocytes results in firm integrin-mediated binding of the leukocytes to the

endothelium at the site of infection. The leukocytes stop rolling, their cytoskeleton is

reorganized, and they spread out

on the endothelial surface.

The next step in the process is migration of the leukocytes through the endothelium, called

transmigration or diapedesis. Chemokines act on the adherent leukocytes and stimulate the

cells to migrate through interendothelial spaces toward the chemical concentration gradient,

that is, toward the site of injury or infection. Certain homophilic adhesion molecules (i.e.,

adhesion molecules that bind to each other) present in the intercellular junction of

endothelium are involved the migration of leukocytes.

One of these molecules is a member of the immunoglobulin superfamily called PECAM-1

(platelet endothelial cell adhesion molecule) or CD31. Leukocyte diapedesis, similar to

increased vascular

permeability, occurs predominantly in the venules (except in the lungs, where it also occurs

in capillaries). After traversing the endothelium, leukocytes are transiently retarded in their

journey by the continuous basement membrane of the venules, but eventually the cells pierce

the basement membrane, probably by secreting collagenases.

The net result of this process is that leukocytes rapidly accumulate where they

are needed.Once leukocytes enter the extravascular connective tissue, they are able to adhere

to the extracellular matrix by virtue of B1 , integrins and CD44 binding to matrix proteins.

Thus, the leukocytes are retained at the site where they are needed.

The type of emigrating leukocyte varies with the age of the inflammatory response and with

the type of stimulus. In most forms of acute inflammation, neutrophils predominate in the

inflammatory infiltrate during the first 6 to 24 hours, then are replaced by monocytes in 24 to

48 hours .

Neutrophils are more numerous in the blood, they respond more rapidly to chemokines, and

they may attach more firmly to the adhesion molecules that are rapidly induced on

endothelial cells, such as P- and E-selectins. In addition, after entering tissues, neutrophils are

short-lived; they undergo apoptosis and disappear after 24 to 48 hours, whereas monocytes

survive longer.

Chemotaxis

After extravasation, leukocytes emigrate in tissues toward the site of injury by a process

called chemotaxis, defined most simply as locomotion oriented along a chemical gradient. All

granulocytes, monocytes and, to a lesser extent, lymphocytes respond to chemotactic stimuli

with varying rates of speed.

Both exogenous and endogenous substances can act as chemoattractants. The most common

exogenous agents are bacterial products. Some of these are peptides that possess an N-

formyl-methionine terminal amino acid. Others are lipid in nature. Endogenous

chemoattractants, several chemical mediators: (1) components of the complement system,

particularly C5a; (2) products of the lipoxygenase pathway, mainly leukotriene B4 (LTB 4 );

and (3) cytokines, particularly those of the chemokine family (e.g.,

IL-8).

All the chemotactic agents mentioned bind to specific seven transmembrane G-protein–

coupled receptors (GPCRs) on the surface of leukocytes. Signals initiated from these

receptors result in recruitment of G-proteins and activation of several effector molecules,

including phospholipase C (PLCy) and phosphoinositol-3 kinase (P13K), as well as protein

tyrosine kinases. PLCy and PI3K act on membrane inositol phospholipids to generate lipid

second messengers that increase cytosolic calcium and activate small GTPases of the

Rac/Rho/cdc42 family as well as numerous kinases. The GTPases induce polymerization of

actin, resulting in increased amounts of polymerized actin at the leading edge of the cell. The

leukocyte moves by extending filopodia that pull the back of the cell in the direction of

extension, much as an automobile with front-wheel drive is pulled by the wheels in front .

Actin reorganization may also occur

at the trailing edge of the cell. Locomotion involves rapid assembly of actin monomers into

linear polymers at the filopodium's leading edge, followed by cross-linking of filaments, and

disassembly of such filaments away from the leading edge.

A number of actin-regulating proteins, such as filamin, gelsolin, profilin, and calmodulin,

interact with actin

and myosin in the filopodium to produce contraction.

Leukocyte Activation

Microbes, products of necrotic cells, antigen-antibody complexes, and cytokines, including

chemotactic factors, induce a number of responses in leukocytes that are part of the defensive

functions of the leukocytes (neutrophils and monocytes/ macrophages) and are referred to

under the rubric of leukocyte activation. Activation results from several signaling pathways

that are triggered in leukocytes, resulting in increases in cytosolic Ca2+ and activation of

enzymes such as protein kinase C and phospholipase A2 .

The functional responses that are induced on leukocyte activation include the following:

■ Production of arachidonic acid metabolites from phospholipids, as a result of activation of

phospholipase A2, by increased intracellular calcium and other signals.

■ Degranulation and secretion of lysosomal enzymes and activation of the oxidative burst

(discussed below under phagocytosis).

■ Secretion of cytokines, which amplify and regulate inflammatory reactions. Activated

macrophages are the chief source of the cytokines that are involved in inflammation, but mast

cells and other leukocytes may contribute.

■ Modulation of leukocyte adhesion molecules.,different cytokines cause increased

endothelial expression of adhesion molecules and increased avidity of leukocyte integrins,

allowing firm adhesion of activated neutrophils to endothelium.

Phagocytosis

Phagocytosis is the process by which cells ingest particles of a size visible to light

microscopy. Neutrophils and monocytes/macrophages are the only cells efficient enough at

phagocytosis to be considered "professional phagocytes." Phagocytosis results in the eventual

containment of a pathogen within a membrane-delimited structure, the phagosome The

immune system has evolved mechanisms of coating the

pathogen with a few recognizable ligands, which enable the phagocyte to bind and ingest the

pathogen. This is referred to as opsonization.

Once a microbe has been ingested, it may be killed. Phagocytes kill bacteria by two broad

categories of killing mechanisms. One category is based on the reduction of oxygen and is

referred to as oxidative.

Oxidative mechanisms require 1) the presence of oxygen and 2) an oxidation-reduction

potential, Eh, at or above -160 mV. Both variables may be suboptimal within the gingival

crevice. Neutrophils do not require oxygen for energy and can function under anaerobic

conditions. Thus phagocytes also must possess the second category of killing mechanisms,

the nonoxidative mechanisms.

Nonoxidative killing requires phagosome-lysosomefusion. This process involves the

movement toward and subsequent membrane fusion of the lysosome with the phagosome,

forming a phagolysosome. This results in

the secretion of lysosomal components into the phagolysosome. Each neutrophil possesses

two main types of lysosomes, or granules: specific granules, designed for both extracellular

and intraphagolysosomal secretion, and azurophil granules, designed mainly for

intraphagolysosomal secretion. Less than 30 seconds after phagocy tosis, neutrophils secrete

specific granule components into the phagolysosome. Specific granules contain several

microbiocidal components including lysozyme and lactoferrin.

Lysozyme is an enzyme that possesses enzyme dependent bactericidal activity and enzyme-

independent

bactericidal and fungicidal activity. Lactoferrin is a bacteriostatic compound that contains a

bactericidal peptide domain, lactoferricin. Neutrophils secrete azurophil granule components

into the phagolysosome minutes after the secretion of the specific granules. Among the

microbicidal compounds are small antimicrobial peptides known as a-defensins (e.g., HNP-1,

HNP-2, HNP- 3, and HNP-4), serprocidins (elastase, proteinase 3,azurocidin), cathepsin G,

and lysozyme.

These nonoxidative mechanisms of neutrophil killing may be of particular importance in

periodontal diseases because of the highly anaerobic conditions in the subgingival

environment.

In the presence of oxygen, phagocytes additionally possess mechanisms of oxidative killing.

In particular, neutrophils exert intense microbicidal activity by forming toxic, reduced

oxygen metabolites such as superoxide anion (O2) using the NADPH oxidase system. The

superoxide anion also contributes to the formation of hydrogen peroxide (H2O2), which is

capable of diffusing across membranes. Inside a target cell, H 2O2 may be further reduced to

the hydroxyl radical, which can cause DNA damage. More importantly, H202 is a substrate

for myeloperoxidase (WO). In the presence of H2O2 and chloride, MPO catalyzes the

formation of hypochlorous acid (HOCI). This molecule is the acidic form of the laundry

bleach salt, sodium hypochlorite (NaOCI), which also is used as an antimicrobialcleansing

irrigant in endodontics.

Deficiencies in the NADPH oxidase system result in chronic granulomatous disease, a severe,

recurrent, focal infection by organisms which do not release H 2O2 on their own. Chronic

granulomatous disease has been associated inconsistently with aggressive periodontal

disease, suggesting that oxidative microbicidal mechanisms are of some importance in

periodontal infections.

In summary, phagocytosis is of primary importance in the ability of a host to resist or combat

infection.

Once a pathogenic microorganism is ingested, several mechanisms of killing are possible.

Because of the highly anaerobic conditions in the periodontal environment, nonoxidative

mechanisms of killing are of particular importance.

I. CHEMICAL MEDIATORS OF INFLAMMATION

Also called as permeability factor or endogenous mediators of increased vascular

permeability. These are endogenous compounds that direct the vascular and cellular

events in inflammation.

Broadly classified into –

Mediators released by cells

Mediators originating from plasma

Cell - derived mediators

1. Vaso active amines [Histamine, 5 – HT]

2. Arachidonic acid metabolites

a) Metabolites via cyclo–oxygenase pathway [prostaglandins,

thromboxane A2, protacyclin]

b) Metabolites via lipo – oxygenase pathway [5 – HETE, Leukotrienes]

3. Lysosomal components

4. Platelet activating factor

5. Cytokines [IL – 1, TNF – α, β , IFN –γ, chemokines]

Nitric oxide and oxygen metabolites

VASOACTIVE AMINES

The two amines, histamine and serotonin, are especially important because they are present in

preformed stores in cells and are therefore among the first mediators to be released during

inflammation.

Histamine

Histamine is widely distributed in tissues, the richest source being the mast cells that are

normally present in the connective tissue adjacent to blood vessels. It is also found in blood

basophils and platelets. Preformed histamine is present in mast cell granules and is released

by mast cell degranulation in response to a variety of stimuli: (1) physical injury such as

trauma, cold, or heat; (2) immune reactions involving binding of antibodies to mast cells ;

C5a); (4) histamine-releasing proteins derived from leukocytes; (5) neuropeptides (e.g.,

substance P); and (6) cytokines (IL-1, IL-8).

In humans, histamine causes dilation of the arterioles and increases the permeability of

venules (it, however, constricts large arteries). It is considered to be the principal mediator of

the immediate transient phase of increased vascular permeability, causing venular gaps, as we

have seen. It acts on the microcirculation mainly via binding to H, receptors on endothelial

cells.

Serotonin

Serotonin (5-hydroxytryptamine) is a preformed vasoactive mediator with actions similar to

those of histamine. It is present in platelets and enterochromaffin cells, and in mast cells in

rodents but not humans.

Release of serotonin (and histamine) from platelets is stimulated when platelets aggregate

after contact with collagen, thrombin, adenosine diphosphate (ADP), and antigenantibody

complexes. Platelet aggregation and release are also stimulated by platelet activating factors

(PAF) derived from mast cells during IgE-mediated reactions. In this way, the platelet release

reaction results in increased permeability

during immunologic reactions.

PLATELET ACTIVATING FACTOR (PAF)

PAF is another bioactive phospholipid-derived mediator. Its name comes from its initial

discovery as a factor derived from antigen-stimulated, IgE-sensitized basophils that causes

platelet aggregation, but it is now known to have multiple inflammatory effects. Chemically,

PAF is acetyl-glycerylether phosphorylcholine (AGEPC), a phospholipid with a typical

glycerol backbone, a long-chain fatty acid in the A position, an unusually short chain

substituent in the B location, and a phosphatidylcholine moiety.

PAF mediates its effects via a single G-protein-coupled receptor, and its effects are regulated

by a family of inactivating PAF acetylhydrolases. A variety of cell types, including platelets,

basophils (and mast cells), neutrophils, monocytes/ macrophages, and endothelial cells, can

elaborate PAF, in both secreted and cell-bound forms.

In addition to platelet stimulation, PAF causes vasoconstriction and bronchoconstriction, and

at extremely low concentrations it induces vasodilation and increased venular permeability

with a potency 100 to 10,000 times greater than that of histamine.

PAF also causes increased leukocyte adhesion to endothelium (by enhancing integrin-

mediated leukocyte binding), chemotaxis, degranulation, and the oxidative burst. Thus, PAF

can elicit most of the cardinal features of inflammation. PAF also boosts the synthesis of

other mediators, particularly eicosanoids, by leukocytes and other cells.

A role for PAF in vivo is supported by the ability of synthetic PAF receptor antagonists to

inhibit inflammation in some experimental models. There are as yet no drugs approved for

clinical use

that function as specific PAF antagonists.

ARACHIDONIC ACID METABOLITES: PROSTAGLANDINS, LEUKOTRIENES,

AND

LIPOXINS

When cells are activated by diverse stimuli, their membrane lipids are rapidly remodeled to

generate biologically active lipid mediators that serve as intracellular or extracellular signals

to affect a variety of biologic processes, including inflammation and hemostasis. These lipid

mediators are thought of as autocoids, or short-range hormones that are formed rapidly, exert

their effects locally, and then either decay spontaneously or are destroyed enzymatically.

Arachidonic acid (AA) is a 20-carbon polyunsaturated fatty acid (5,8,11,14-eicosatetraenoic

acid) that is derived from dietary sources or by conversion from the essential fatty acid

linoleic acid. It does not occur free in the cell but is normally esterified in membrane

phospholipids. It is released from membrane phospholipids through the action of cellular

phospholipases (e.g., phospholipase A2), which may be activated by mechanical, chemical,

and physical stimuli or by other mediators (e.g., C5a).

The biochemical signals involved in the activation of phospholipase A 2 include an increase

in cytoplasmic

Ca2+ and activation of various kinases in response to external stimuli. AA metabolites, also

called eicosanoids, are synthesized by two major classes of enzymes: cyclooxygenases

(prostaglandins and thromboxanes) and lipoxygenases (leukotrienes and lipoxins) .

Eicosanoids bind to G protein–coupled receptors on many cell types and can mediate

virtually every step of inflammation . They can be found in inflammatory exudates, and their

synthesis is increased at sites of inflammation. Structurally distinct agents that suppress

cyclooxygenase activity (aspirin, nonsteroidal

anti-inflammatory drugs [NSAIDs], and COX-2 inhibitors'`) reduce inflammation in vivo.

The cyclooxygenases and lipoxygenase produce different mediators from the AA precursor.

The cyclooxgenase pathway, initiated by two different enzymes (the constitutively expressed

COX-1 and the inducible enzyme COX-2), leads to the generation of prostaglandins.

Prostaglandins are divided into series based on structural features as coded by a letter (PGD,

PGE, PGF, PGG, and PGH) and a subscript numeral (e.g., 1, 2), which indicates the number

of double bonds in the compound.

The most important ones in inflammation are PGE 2, PGD 2 , PGF2, PGI2 (prostacyclin),

and TxA2 (thromboxane), each of which is derived by the action of a specific enzyme on an

intermediate in the pathway. Some of these enzymes have restricted tissue distribution. For

example, platelets contain the enzyme thromboxane synthetase, and hence TxA2 is the major

product in these cells. TxA 2 , a potent plateletaggregating agent and vasoconstrictor, is itself

unstable and rapidly converted to its inactive form TXB2. Vascular endothelium lacks

thromboxane synthetase but possesses prostacyclin synthetase, which leads to the formation

of prostacyclin (PGI 2 ) and its stable end product PGF1a.

Prostacyclin is a vasodilator, a potent inhibitor of platelet aggregation, and also markedly

potentiates the

permeability-increasing and chemotactic effects of other mediators. A thromboxane–

prostacyclin imbalance has been implicated as an early event in thrombus formation in

coronary and cerebral blood vessels.

The prostaglandins are also involved in the pathogenesis of pain and fever in inflammation.

PGE 2 is hyperalgesic in that it makes the skin hypersensitive to painful stimuli. It causes a

marked increase in pain produced by intradermal injection of suboptimal concentrations of

histamine and bradykinin and is involved in cytokine-induced fever during infections . PGD 2

is the major metabolite of the cyclooxygenase pathway in mast cells; along with PGE 2 and

PGF,,, (which are more widely distributed), it causes vasodilation and increases the

permeability of postcapillary venules, thus potentiating edema formation.

There has been great interest in the COX-2 enzyme because it is induced by a variety of

inflammatory stimuli and is absent in most tissues under normal "resting" conditions

.

COX-1, by contrast, is produced in response to inflammatory stimuli and is also

constitutively expressed in

most tissues. This difference has led to the notion that COX- 1 is responsible for the

production of prostaglandins that are involved in inflammation but also serve a homeostatic

function (e.g., fluid and electrolyte balance in the kidneys, cytoprotection in the

gastrointestinal tract). In contrast, COX-2

stimulates the production of the prostaglandins that are involved in inflammatory reactions

.

In the lipoxygenase pathway, the initial products are generated by three different

lipoxygenases, which are

present in only a few types of cells. 5-lipoxygenase (5-LO) is the predominant enzyme in

neutrophils. The main product, 5-HETE, which is chemotactic for neutrophils, is converted

into a family of compounds collectively called leukotrienes. LTB, is a potent chemotactic

agent and activator of neutrophil functional responses, such as aggregation and adhesion of

leukocytes to venular endothelium, generation of oxygen free radicals, and release of

lysosomal enzymes. The cysteinyl-containing leukotrienes C„ D„ and E, (LTC„ LTD,, and

LTE,) cause intense vasoconstriction, bronchospasm, and increased vascular permeability.

The vascular leakage, as with histamine, is restricted to venules. Leukotrienes are several

orders of magnitude more potent than histamine in increasing vascular permeability and

causing bronchospasm. Leukotrienes mediate their actions by binding to cystein leukotreine 1

(CysLTI) and CysLT2 receptors. They are important in the pathogenesis of bronchial asthma.

Lipoxins are a recent addition to the family of bioactive products generated from AA, and

transcellular biosynthetic mechanisms (involving two cell populations) are key to their

production. Leukocytes, particularly neutrophils, produce intermediates in lipoxin synthesis,

and these are converted to lipoxins by platelets interacting with the leukocytes. Lipoxins A4

and B 4 (LXA„ LXB4 ) are generated by the action of platelet 12-lipoxygenase on neutrophil

derived LTA. Cell-cell contact enhances transcellularmetabolism, and blocking adhesion

inhibits lipoxin production. The principal actions of lipoxins are to inhibit

leukocyte recruitment and the cellular components of inflammation.

They inhibit neutrophil chemotaxis and adhesion to endothelium. There is an inverse

relationship between the amount of lipoxin and leukotrienes formed, suggesting that the

lipoxins may be endogenous negative regulators of leukotriene action and may thus play a

role in the resolution of inflammation.

A new class of arachidonic acid-derived mediators, called resolvins, have been identified in

experimental animals treated with aspirin. These mediators inhibit leukocyte recruitment and

activation, in part by inhibiting the production of cytokines. Thus, the anti-inflammatory

activity of aspirin is likely attributable to its ability to inhibit cyclooxygenases and, perhaps,

to stimulate the production of resolvins.

Anti-inflammatory therapy can be directed at many targets along the eicosanoid biosynthetic

pathways:

Cyclooxygenase inhibitors include aspirin and other nonsteroidal anti-inflammatory drugs

( NSAIDs), such as indomethacin. They function by inhibiting prostaglandin synthesis;

aspirin does this by irreversibly acetylating and inhibiting cyclooxygenase. COX-2 inhibitors

are a newer class of these drugs. The finding that COX-2 is inducibly expressed only in

response to inflammatory stimuli was the impetus for developing antagonists against this

enzyme to reduce inflammation without interfering with the physiologic functions of AA

metabolites. COX-2 inhibitors are now widely used as anti-inflammatory drugs and generally

produce less toxicity than the older COX-1 inhibitors.

Lipoxygenase inhibitors. 5-lipoxygenase is not affected by NSAIDs, and many new inhibitors

of this enzyme pathway have been developed. Pharmacologic agents that inhibit leukotriene

production or block leukotriene receptors (CysLT1 and CysLT2) have been found useful in

the treatment of asthma.

Broad-spectrum inhibitors include glucocorticoids. These powerful anti-inflammatory

agents may act by downregulating the expression of specific target genes, including the genes

encoding COX-2, phospholipase A2 proinflammatory cytokines (such as IL-1 and TNF), and

nitric oxide synthase (iNOS) . Glucocorticoids also upregulate genes that encode potent anti-

inflammatory proteins, such as lipocortin 1. Lipocortin 1 inhibits release of AAphospholipids

from membrane

CYTOKINES AND CHEMOKINES

Cytokines are proteins produced by many cell types (principally activated lymphocytes and

macrophages, but also endothelium, epithelium, and connective tissue cells) that modulate the

functions of other cell types. Long known to be involved in cellular immune responses, these

products have additional effects that play important roles in both acute and chronic

inflammation

Tumor Necrosis Factor and Interleukin-1

TNF and IL-I are two of the major cytokines that mediate inflammation. They are produced

mainly by activated macrophages. A cytokine resembling TNF, called lymphotoxin

(previously called TNF-13, to distinguish it from TNF, which was called TNF-a), is produced

by activated T lymphocytes, and IL-1 may be produced by many other cell types as well.

The secretion of TNF and IL-1 can be stimulated by endotoxin and other microbial products,

immune complexes, physical injury, and a variety of inflammatory stimuli. Their most

important actions in inflammation are their effects on endothelium, leukocytes, and

fibroblasts, and induction of systemic acute-phase reactions.

In endothelium, they induce a spectrum of changes—mostly regulated at the level of gene

transcription—referred to as endothelial activation. In particular, they induce the synthesis of

endothelial adhesion molecules and chemical mediators, including other cytokines,

chemokines, growth factors, eicosanoids, and nitric oxide (NO); production of enzymes

associated with matrix remodeling; and increases in the surface thrombogenicity of the

endothelium. TNF also induces priming of neutrophils, leading to augmented responses of

these cells to other mediators.

IL-1 and TNF (as well as IL-6) induce the systemic acutephase responses associated with

infection or injury. Features of these systemic responses include fever, loss of appetite,

slowwave sleep, the release of neutrophils into the circulation, the release of corticotropin

and corticosteroids and, particularly with regard to TNF, the hemodynamic effects of septic

shock—hypotension, decreased vascular resistance, increased heart rate, and decreased blood

pH .

TNF also regulates body mass by promoting lipid and protein mobilization and by

suppressing appetite. Sustained production of TNF contributes to cachexia, a pathologic state

characterized by weight loss and anorexia that accompanies some infections and neoplastic

diseases.

Chemokines

Chemokines are a family of small (8 to 10 kD) proteins that act primarily as chemoattractants

for specific types of leukocytes. About 40 different chemokines and 20 different receptors for

chemokines have been identified. They are classified into four major groups, according to the

arrangement of the conserved cysteine (C) residues in the mature proteins:

C-X-C chemokines (also called a chemokines) have one amino acid residue separating the

first two conserved cysteine residues. The C-X-C chemokines act primarily on neutrophils.

IL-8 is typical of this group. It is secreted by activated macrophages, endothelial cells, and

other cell types and causes activation and chemotaxis of neutrophils, with limited activity on

monocytes and eosinophils. Its most important inducers are microbial products and other

cytokines, mainly IL-1 and TNF.

C-C chemokines (also called 13 chemokines) have the first two conserved cysteine residues

adjacent. The C-C chemokines, which include monocyte chemoattractant protein (MCP-1),

eotaxin, macrophage inflammatory protein-1a (MIP-la), and RANTES (regulated and normal

T cell expressed and secreted), generally attract monocytes, eosinophils, basophils, and

lymphocytes but not neutrophils.

Although most of the chemokines in this class have overlapping properties, eotaxin

selectively recruits

eosinophils.

C chemokines (also called y chemokines) lack two (the first and third) of the four conserved

cysteines. The C chemokines (e.g., lymphotactin) are relatively specific for lymphocytes.

CX3C chemokines contain three amino acids between the two cysteines. The only known

member of this class is called fractalkine. This chemokine exists in two forms: the cell

surface-bound protein can be induced on endothelial cells by inflammatory cytokines and

promotes strong adhesion of monocytes and T cells, and a soluble form, derived by

proteolysis of the membrane-bound protein, has potent chemoattractant activity for the same

cells.

Chemokines mediate their activities by binding to seven transmembrane G-protein-coupled

receptors. These receptors (called CXCR or CCR, for C-X-C or C-C chemokine receptors)

usually exhibit overlapping ligand specificities, and leukocytes generally express more than

one receptor type.Certain chemokine receptors

(CXCR-4, CCR-5) act as coreceptors for a viral envelope glycoprotein of human

immunodeficiency virus (HIV-1) and are thus involved in binding and entry of the virus into

cells.

Chemokines stimulate leukocyte recruitment in inflammation and control the normal

migration of cells through various tissues. Some chemokines are produced transiently in

response to inflammatory stimuli and promote the recruitment of leukocytes to the sites of

inflammation. Other chemokines are produced constitutively in tissues and function in

organogenesis to organize different cell types in different anatomic regions of the tissues. In

both situations, chemokines may be displayed at high concentrations attached

to proteoglycans on the surface of endothelial cells and in the extracellular matrix.

NITRIC OXIDE (NO)

NO, a pleiotropic mediator of inflammation, was discovered as a factor released from

endothelial cells that caused vasodilation by relaxing vascular smooth muscle and was

therefore called endothelium-derived relaxing factor.

NO is a soluble gas that is produced not only by endothelial cells, but also by macrophages

and some neurons in the brain. NO acts in a paracrine manner on target cells through

induction of cyclic guanosine monophosphate (GMP), which, in turn, initiates a series of

intracellular events leading to a response, such

as the relaxation of vascular smooth muscle cells. Since the in vivo half-life of NO is only

seconds, the gas acts only on cells in close proximity to where it is produced.

NO is synthesized from L-arginine by the enzyme nitric oxide synthase (NOS). There are three

different types of NOS— endothelial (eNOS), neuronal (nNOS), and inducible (iNOS)—

which exhibit two patterns of expression. eNOS and nNOS are constitutively expressed at low

levels and can be activated rapidly by an increase in cytoplasmic calcium ions. Influx of

calcium into cells leads to a rapid production of NO.

iNOS, in contrast, is induced when macrophages and other cells are activated by cytokines

(e.g., TNF, IFN-y) or other agents

NO and its derivatives are microbicidal, and thus NO is also a mediator of host defense

against infection. Evidence supporting the importance of this antimicrobial activity of NO

includes the following: (1) reactive nitrogen intermediates derived from NO possess

antimicrobial activity; (2) interactions occur between NO and reactive oxygen intermediates,

leading to the formation of multiple antimicrobial metabolites; (3) production of NO is

increased during host responses to infection; and (4) genetic inactivation of iNOS enhances

microbial replication in experimental animal models. High levels of NO production by a

variety of cells appear to limit the replication of bacteria, helminths, protozoa, and viruses (as

well as tumor cells).

LYSOSOMAL CONSTITUENTS OF LEUKOCYTES

Neutrophils and monocytes contain lysosomal granules, which, when released, may

contribute to the inflammatory response. Neutrophils have two main types of granules. The

smaller specific (or secondary) granules contain lysozyme, collagenase, gelatinase,

lactoferrin, plasminogen activator, histaminase, and alkaline phosphatase. The large

azurophil (or primary) granules contain myeloperoxidase, bactericidal factors (lysozyme,

defensins), acid hydrolases, and a variety of neutral proteases (elastase, cathepsin G,

nonspecific collagenases, proteinase ).

Both types of granules can empty into phagocytic vacuoles that form around engulfed

material, or the granule contents can be released into the extracellular space. The specific

granules are secreted extracellularly more readily and by lower concentrations of agonists,

whereas the potentially more destructive azurophil granules release their contents primarily

within the phagosonme and require high levels of agonists to be released extracellularly.

Different granule enzymes serve different functions. Acid proteases degrade bacteria and

debris within the phagolysosomes, in which an acid pH is readily reached. Neutral proteases

are capable of degrading various extracellular components. These enzymes can attack

collagen, basement membrane, fibrin, elastin, and cartilage, resulting in the tissue destruction

that accompanies inflammatory processes.

Neutral proteases can also cleave C3 and C5 directly, releasing anaphylatoxins, and release a

kinin-like peptide from kininogen. Neutrophil elastase has been shown to degrade virulence

factors of bacteria and thus combat bacterial infections.

Monocytes and macrophages also contain acid hydrolases, collagenase, elastase,

phospholipase, and plasminogen activator. These may be particularly active in chronic

inflammatory reactions. Because of the destructive effects of lysosomal enzymes, the initial

leukocytic infiltration, if unchecked, can potentiate further increases in vascular permeability

and tissue damage. These harmful proteases, however, are held in check by a system of

antiproteases in the serum and tissue fluids. Foremost among these is a,-antitrypsin, which is

the major inhibitor of neutrophil elastase. A deficiency of these inhibitors may lead to

sustained action of leukocyte proteases, as is the case in patients with a1-antitrypsin

deficiency. a-Macroglobulin is another antiprotease found in serum and various secretions.

OXYGEN-DERIVED FREE RADICALS

Oxygen-derived free radicals may be released extracellularly from leukocytes after exposure

to microbes, chemokines, and immune complexes, or following a phagocytic challenge. Their

production is dependent, on the activation of the NADPH oxidative system. Superoxide anion

( 0,), hydrogen peroxide (H2O2 ), and hydroxyl radical (OH) are the major species produced

within the cell, and these metabolites can combine with NO to form other reactive nitrogen

intermediates. Extracellular release of low levels of these potent mediators can increase the

expression of chemokines (e.g., IL-8), cytokines, and endothelial leukocyte adhesion

molecules, amplifying the cascade that elicits the inflammatory response. The physiologic

function of these reactive oxygen intermediates is to destroy phagocytosed microbes. At

higher levels, release of these potent mediators can be damaging to the host. They are

implicated in the following responses:

a) Endothelial cell damage, with resultant increased vascular permeability. Adherent

neutrophils, when activated, not only produce their own toxic species, but also stimulate

xanthine oxidation in endothelial cells themselves, thus elaborating more superoxide.

b) Inactivation of antiproteases, such as a,-antitrypsin. This leads to unopposed protease

activity, with increased destruction of extracellular matrix.

c) Injury to other cell types (parenchymal cells, red blood cells).

II. PLASMA DERIVED MEDIATORS (PLASMA PROTEASES)

These include the various products derived from activation and interaction of 4

interlinked systems: kinin, fibrinolytic, clotting and complement

Complement System

The complement system consists of 20 component proteins (and their cleavage products),

which are found in greatest concentration in plasma. This system functions in both innate and

adaptive immunity for defense against microbial agents. In the process of complement

activation, a number of complement components are elaborated that cause increased vascular

permeability, chemotaxis, and opsonization.

Complement proteins are present as inactive forms in plasma and are numbered Cl through

C9.Many of these proteins are activated to become proteolytic enzymes that degrade other

complement proteins, thus forming a cascade capable of tremendous enzymatic amplification.

The critical step in the elaboration of the biologic functions of complement is the activation

of the third (and most abundant) component, C3.

Cleavage of C3 can occur by one of three pathways: the classical pathway, which is triggered

by fixation of Cl to antibody (IgM or IgG) combined with antigen; the alternative pathway,

which can be triggered by microbial surface molecules (e.g., endotoxin, or LPS), complex

polysaccharides, cobra venom,

and other substances, in the absence of antibody; and the lectin pathway, in which plasma

mannose-binding lectin binds to carbohydrates on microbes and directly activates Cl.

Whichever pathway is involved in the early steps of complement activation, they all lead to

the formation of an active enzyme called the C3 convertase, which splits C3 into two

functionally distinct fragments, C3a and C3b. C3a is released and C3b becomes covalently

attached to the cell or molecule where complement is being activated. C3b then binds to the

previously generated fragments to form C5 convertase, which

cleaves C5 to release C5a. The remaining C5b binds the late components (C6-C9),

culminating in the formation of the membrane attack complex (MAC, composed of multiple

C9 molecules).

The biologic functions of the complement system fall into two general categories: cell lysis

by the MAC, and the effects of proteolytic fragments of complement. Complement derived

factors mediate a variety of phenomena in acute inflammation:

a) Vascular phenomena. C3a, C5a, and, to a lesser extent, C4a are split products of the

corresponding complement components that stimulate histamine release from mast cells

and thereby increase vascular permeability and cause vasodilation. They are called

anaphylatoxins because they have effects similar to those of mast cell mediators that are

involved in the reaction called anaphylaxis . C5a also activates the lipoxygenase pathway

of arachidonic acid (AA) metabolism in neutrophils and monocytes, causing further

release of inflammatory mediators.

b) Leukocyte adhesion, chemotaxis, and activation. C5a is a powerful chemotactic agent for

neutrophils, monocytes, eosinophils, and hasophils.

c) Phagocytosis. C3b and its cleavage product iC3b (inactive C3b), when fixed to the

bacterial cell wall, act as opsonins and favor phagocytosis by neutrophils and

macrophages, which bear cell surface receptors for these complement fragments. Among

the complement components, C3 and C5 are the most important inflammatory mediators.

In addition, C3 and C5 can be activated several proteolytic enzymes present within the

inflammatory exudate. These include plasmin and lysosomal enzymes released from

neutrophils .

Thus, the chemotactic effect of complement and the complement-activating effects of

neutrophils can set up a self-perpetuating cycle of neutrophil emigration. The activation of

complement is tightly controlled by cell associated and circulating regulatory proteins.The

presence of these inhibitors in host cell membranes protects the host from inappropriate

damage during protective reactions against microbes.

Kinin System

The kinin system generates vasoactive peptides from plasma proteins, called kininogens, by

the action of specific proteases called kallikreins. Activation of the kinin system results in the

release of the vasoactive nonapeptide bradykinin. Bradykinin increases vascular permeability

and causes contraction of smooth muscle, dilation of blood vessels, and pain when injected

into the skin. These effects are similar to those of histamine. It is triggered by activation of

Hageman factor (factor XII of the intrinsic clotting pathway; see later) upon contact with

negatively charged surfaces, such as collagen and basement membranes. A fragment of factor

XII (prekallikrein activator, or factor XIIa) is produced, and this converts plasma

prekallikrein into an active proteolytic form, the enzyme kallikrein.

The latter cleaves a plasma glycoprotein precursor, high-molecular-weight kininogen, to

produce bradykinin. High-molecular-weight kininogen also acts as a cofactor or catalyst in

the activation of Hageman factor. The action of bradykinin is short-lived because it is quickly

inactivated by an enzyme called kininase. Any remaining kinin is inactivated during passage

of plasma through the lung by angiotensin converting enzyme. Kallikrein itself is a potent

activator of Hageman factor, allowing for autocatalytic amplification of the initial stimulus.

Kallikrein has chemotactic activity, and it also directly converts C5 to the chemoattractant

product C5a.

Clotting System

The clotting system and inflammation are intimately connected processes. The clotting

system is divided into two pathways that converge, culminating in the activation of thrombin

and the formation of fibrin. The

intrinsic clotting pathway is a series of plasma proteins that can be activated by Hageman

factor ( factor XII), a protein synthesized by the liver that circulates in an inactive form until

it encounters collagen or basement membrane or activated platelets (as occurs at the site of

endothelial injury). Factor XII then undergoes a conformational change (becoming factor

Xlfa), exposing an active serine center that can subsequently cleave protein substrates and

activate a variety of mediator systems .

The protease thrombin provides the main link between the coagulation system and

inflammation. Activation of the clotting system results in the activation of thrombin (factor

IIa) from precursor prothrombin (factor II). Thrombin is the enzyme that cleaves circulating

soluble fibrinogen to generate an insoluble fibrin clot and is the major coagulation protease.

It binds to receptors that are called protease-activated receptors (PARs) because they bind

multiple trypsin-like serine proteases in addition to thrombin. These receptors are seven-

transmembrane G protein—coupled receptors that are expressed on platelets, endothelial and

smooth muscle cells, and many other cell types. Engagement of the so-called type 1 receptor

(PAR-1) by proteases, particularly thrombin, triggers

several responses that induce inflammation. They include mobilization of P-selectin,

production of chemokines, and expression of endothelial adhesion molecules for leukocyte

integrins; induction of cyclooxygenase-2 and production of Prostaglandins; production of

PAF and nitric oxide; and changes in endothelial shape.

At the same time that factor XIIa is inducing clotting, it can also activate the fibrinolytic

system. This cascade counterbalances clotting by cleaving fibrin, thereby solubilizing the

fibrin clot. The fibrinolytic system contributes to the vascular phenomena of inflammation in

several ways. Plasminogen activator

(released from endothelium, leukocytes, and other tissues) cleaves plasminogen, a plasma

protein that binds to the evolving fibrin clot to generate plasmin, a multifunctional protease.

Plasmin is important in lysing fibrin clots, but in the context of inflammation it also cleaves

C3 to produce C3 fragments, and it degrades fibrin to form fibrin split products, which may

have permeability-inducing properties. Plasmin

can also activate Hageman factor, which can trigger multiple cascades , amplifying the

response.

Thus the following conclusions can be drawn:

a)Bradykinin, C3a, and C5a (as mediators of increased vascular permeability); C5a (as the

mediator of chemotaxis); and thrombin (which has effects on endothelial and many other cell

types) are likely to be the most important in vivo.

b)C3a and C5a can be generated by several types of reactions: (1) immunologic reactions,

involving antibodies and complement (the classical pathway); (2) activation of the alternative

or lectin complement pathways by microbes, in the absence of antibodies; and (3) agents not

directly related to immune responses, such as plasmin, kallikrein, and some serine proteases

found in normal tissue.

c)Activated Hageman factor (factor X IIa) initiates four systems involved in the inflammatory

response:

(1) the kinin system, which produces vasoactive kinins;

(2) the clotting system, which induces formation of thrombin, fibrinopeptides, and factor X,

which have inflammatory properties;

(3) the fibrinolytic system, which produces plasmin and degrades the fibrin; and

(4) the complement system, which produces anaphylatoxins. Some of the products of this

initiation—particularly kallikrein—can, by feedback, activate Hageman factor, resulting in

profound amplification of

the effects of the initial contact.

CHRONIC INFLAMMATION

Chronic inflammation is defined as prolonged process in which tissue destruction and

inflammation occur at the same time.

Chronic inflammation can be caused by one of the following ways-

1) Persistent infections by certain microorganisms, such as tubercle bacilli, Treponema

pallidum (the causative organism of syphilis), and certain viruses, fungi, and parasites. These

organisms are of low toxicity and evoke an immune reaction called delayed type

hypersensitivity.The inflammatory response sometimes takes a specific pattern called a

granulomatous reaction

.

2) Prolonged exposure to potentially toxic agents, either exogenous or endogenous.

3) Autoimmunity. Under certain conditions, immune reactions develop against the individual's

own tissues, leading to autoimmune diseases . In these diseases, autoantigens evoke a self-

perpetuating immune reaction

that results in chronic tissue damage and inflammation. Immune reactions play an important

role in several

common chronic inflammatory diseases, such as rheumatoid arthritis and lupus

erythematosus.

MORPHOLOGIC FEATURES

In contrast to acute inflammation, which is manifested by vascular changes, edema, and

predominantly neutrophilic infiltration, chronic inflammation is characterized by:

■ Infiltration with mononuclear cells, which include macrophages, lymphocytes, and plasma

cells.

■ Tissue destruction, induced by the persistent offending agent or by the inflammatory cells.

■ Attempts at healing by connective tissue replacement of damaged tissue, accomplished by

proliferation of small blood vessels (angiogenesis) and, in particular, fibrosis.

MONONUCLEAR CELL INFILTRATION

Macrophages are one component of the mononuclear phagocyte system . The mononuclear

phagocyte system (sometimes called reticuloendothelial system) consists of closely related

cells of bone marrow origin, including blood monocytes and tissue macrophages. The latter

are diffusely scattered in the connective tissue or located in organs such as the liver (Kupffer

cells), spleen and lymph nodes (sinus histiocytes), and lungs (alveolar macrophages).

Mononuclear phagocytes arise from a common precursor in the bone marrow, which gives

rise to blood monocytes. From the blood, monocytes migrate into various tissues and

differentiate into macrophages. The half-life of blood monocytes is about I day, whereas the

life span of tissue macrophages is several months or years. The journey from bone marrow

stem cell to tissue macrophage is regulated by a variety of growth and differentiation factors,

cytokines, adhesion molecules, and cellular interactions.

Monocytes begin to emigrate into extravascular tissues quite early in acute inflammation, and

within 48 hours they may constitute the predominant cell type. Extravasation of monocytes is

governed by the same factors that are involved in neutrophil emigration, that is, adhesion

molecules and chemical mediators with chemotactic and activating properties. When the

monocyte reaches the extravascular tissue, it undergoes transformation into a larger

phagocytic cell, the macrophage. Macrophages may be activated by a variety of stimuli,

including cytokines (e.g., IFN-y) secreted by sensitized T lymphocytes and by NK cells,

bacterial

endotoxins, and other chemical mediators.

Activation results in increased cell size, increased levels of lysosomal enzymes, more active

metabolism, and greater ability to phagocytose and kill ingested microbes. Activated

macrophages secrete a wide variety of biologically active products that, if unchecked, result

in the tissue injury and fibrosis characteristic of chronic inflammation . In short-lived

inflammation, if the irritant is eliminated, macrophages eventually disappear (either dying off

or making their way into the lymphatics and lymph nodes). In chronic inflammation,

macrophage accumulation persists, and is mediated by different mechanisms :

1. Recruitment of monocytes from the circulation, which results from the expression of

adhesion molecules and chemotactic factors.' Most of the macrophages present in a focus of

chronic inflammation are recruited from circulating monocytes. The process of monocyte

recruitment is fundamentally similar to the recruitment of neutrophils. Chemotactic stimuli

for monocytes include chemokines produced by activated macrophages, lymphocytes, and

other cell types (e.g., MCP- 1); C5a; growth factors such as platelet-derived growth factor

and transforming growth factor-a (TGF-a); fragments from the breakdown of collagen and

fibronectin; and fibrinopeptides. Each of these may play a role under given circumstances; for

example, chemokines are major stimulivfor macrophage accumulation in delayed-

hypersensitivity immune reactions.

2. Local proliferation of macrophages after their emigration from the bloodstream. Once

thought to be an unusualevent, macrophage proliferation is now known to occur prominently

in some chronic inflammatory lesions, such as atheromatous plaques

3. Immobilization of macrophages within the site of inflammation. Certain cytokines and

oxidized lipids can cause such immobilization. The products of activated macrophages serve

to eliminate injurious agents such as microbes and to initiate the process of repair, and are

responsible for much of the tissue injury in chronic inflammation. Some of these products are

toxic to microbes and host cells (e.g., reactive oxygen and nitrogen intermediates) or

extracellular matrix (proteases); some cause influx of other cell types (e.g., cytokines,

chemotactic factors); and still others cause fibroblast proliferation, collagen deposition, and

angiogenesis (e.g., growth factors). This impressive arsenal of mediators makes macrophages

powerful allies in the body's defense against unwanted invaders, but the same weaponry can

also induce considerable tissue destruction when macrophages are inappropriately activated.

Thus, tissue destruction is one of the hallmarks of chronic inflammation

OTHER CELLS IN CHRONIC INFLAMMATION

Other cell types present in chronic inflammation include lymphocytes, plasma cells,

eosinophils, and mast cells:

■ Lymphocytes are mobilized in both antibody-mediated and cell-mediated immune reactions

and even in

nonimmune inflammation. Antigen-stimulated (effector and memory) lymphocytes of

different types (T, B) use various adhesion molecule pairs (predominantly the integrins and

their ligands) and chemokines to migrate into inflammatory sites. Cytokines from activated

macrophages, mainly TNF, IL-1, and chemokines, promote leukocyte recruitment, setting the

stage for persistence of the inflammatory response.

Lymphocytes and macrophages interact in a bidirectional way, and these reactions play an

important role in chronic inflammation .

Macrophages display antigens to T cells, and produce membrane molecules (costimulators)

and cytokines (notably IL-12) that stimulate T-cell responses . Activated T lymphocytes

produce cytokines, and one of these, IFN-y, is a major activator of macrophages.

Plasma cells develop from activated B lymphocytes and produce antibody directed either

against persistent antigen in the inflammatory site or against altered tissue components. In

some strong chronic inflammatory reactions, the accumulation of lymphocytes, antigen-

presenting cells, and plasma cells may assume the morphologic features of lymphoid organs,

particularly lymph nodes, even containing well-formed germinal centers. This pattern of

lymphoid organogenesis is often seen in the synovium of patients with long-standing

rheumatoid arthritis.

■ Eosinophils are abundant in immune reactions mediated by IgE and in parasitic infections .

The recruitment of eosinophils involves extravasation from the blood and their migration into

tissue by processes similar to those for other leukocytes. One of the chemokines that is

especially important for eosinophil recruitment is eotaxin. Eosinophils have granules that

contain major basic protein, a highly cationic protein that is toxic to parasites but also causes

lysis of mammalian epithelial cells. They may thus be of benefit in controlling parasitic

infections but they contribute to tissue damage in immune reactions

■ Mast cells are widely distributed in connective tissues and participate in both acute and

persistent inflammatory reactions. Mast cells express on their surface the receptor that binds

the Fc portion of IgE antibody (FcERI). In acute reactions, IgE antibodies bound to the cells .

Fc receptors specifically recognize antigen, and the cells degranulate and release mediators,

such as histamine and products of AA .This type of response occurs during anaphylactic

reactions to foods, insect venom, or drugs, frequently with catastrophic results. When

properly regulated, this response can benefit the host. Mast cells are also present in chronic

inflammatory reactions, and may produce cytokines that contribute to fibrosis

GINGIVAL INFLAMMATION

Within 10-20 days of plaque accumulation, clinical signs of gingivitis are established

inmost individual although this varies greatly, with some individuals being intrinsically

resistant and other more prone to overt gingivitis (Von der Weijden et al 1994).

In 1976, Page and Shroeder, classified the progression of gingival and periodontal

inflammation on the basis of available clinical and histopathologic evidence. They divided

the progressing lesion into 4 phases

Initial

Early

Established

Advanced

INITIAL LESION

Features

1. Classic vasculitis of vessels subjacent to the JE.

2. Exudation of fluid from gingival sulcus.

3. Increased migration of leukocytes into the JE and gingival sulcus.

4. Presence of serum proteins, especially fibrin extra vascularly.

5. Alteration of the most coronal portion of the JE.

6. Loss of perivascular collagen.

EARLY LESION

Features

1. Accentuation of the features described for the initial lesion.

2. Accumulation of lymphoid cells immediately subjacent to the JE

at the site of acute inflammation

3. Cytopahtic alterations in resident fibroblasts, possibly associated

with interactions with lymphoid cells.

4. Further loss of the collagen fibre net work supporting the

marginal gingiva.

5. Beginning proliferation of the basal cells of the JE.

ESTABLISHED LESION

Features

1. Persistence of the manifestation of acute inflammation.

2. Predominance of plasma cells but without appreciable bone loss.

3. Presence of immunoglobulins extravascularly in the connective tissue and J.E.

4. Combining loss of connective tissue substances noted in the early lesion.

5. Proliferation, apical migration and lateral extension of the JE, early pocket

formation may or may not be present.

ADVANCED LESION

Features

1. Persistence of features described for established lesion.

2. Extension of the lesion into alveolar bone and periodontal ligament with

significant bone loss.

3. Continued loss of collagen subjacent to the pocket epithelium with fibrosis at

more distant sites.

4. Presence of cytopathically altered plasma cells in absence of altered

fibroblasts.

5. Formation of periodontal pockets.

6. Periods of quiescence and exacerbation.

7. Conversion of the bone marrow distant from the lesion into fibrous connective

tissue.

8. Widespread manifestations of inflammatory and immune pathologic tissue

reactions.

Stages of gingivitis

Stage Time

(days)

Blood vessels Junctional and

sulcular

epithelium

Predominate

immune

cells

Collagen Clinical

findings

Initial

lesion

2-4 V. dilation vasculitis Infiltrated by

PMN’s

PMNs Perivascular

loss

Gingival

flow.

Early

lesion

4-7 Vascular

proliferation

Same as stage I

rete peg

formation

atrophic areas

Lymphocytes Increased

loss around

infiltrates

Erythema

B.O.P.

Establishe

d lesion

14-21 Same as stage II and

blood stasis

Same as stage

II but more

advanced

Plasma cells Continued

loss

Changes in

color, size,

texture, etc.

PERIODONTAL INFLAMMATION

Periodontitis is defined as occurring when the “inflammatory process involves the

gingiva and the periodontium resulting in loss of periodontal attachment.

Periodontitis involves loss of clinical attachment and radiographic loss of bone. The

conversion clinically from gingivitis to periodontitis reflects the progression,

histopathologically, from the established stage to advanced stage of the periodontal lesion.

3 plausible hypotheses were put out by Suzuki et al for the progression from gingivitis

to periodontitis.

a. Direct tissue destruction caused by bacterial plaque and metabolic products.

b. Immune hyperresponsiveness precipitated by immune complexes, lymphocyte

blastogenesis or activation of complement pathways.

c. Immune deficiencies involving neutrophil function (chemotaxis,

phagocytosis), neutropenia or autologous mixed lymphocyte response

(AMLR).

CONCLUSION

Inflammation, the commonest of pathological phenomena, is the local tissue reaction to

any one of a number of injurious agents. The number of chemicals that have been

suggested as mediators of acute inflammation is so large, and their inter relationship is so

complex, that it is not possible to give any clear account of their role in inflammation.

The Understanding of various chemical mediators in the progression of periodontal

diseases over the years , have and will decide the future in prevention and treatment of the

same.

REFERENCES

Basic Pathology – Robbins (8th Ed)

Textbook of pathology – Harsh Mohan (4th Ed)

 Inflammation - Andrzej G, Hubert K, Michał Z

Biology Of The Periodontal Tissues - Bartold & Narayanan

Carranza’s Clinical Periodontology - Newman,Takei,Klokkevold,Carranza- 10th

edition

Clinical Periodontology - Jan Lindhe-5th edition

PERIODONTAL WOUND HEALING AND REPAIR

CONTENTS

• Introduction

• Types Of Wound Healing

• Features Of Wound Healing

• Growth Factors In Wound Healing

• Factors Affecting Wound Healing

• Healing After Scaling & Root Planing

• Healing After Surgical Gingivectomy

• Healing After Placement Of Grafts

• Complications of Wound Healing

• Conclusion

• References

INTRODUCTION

WOUND: It is referred to as loss of tissue integrity that leads to disruption of normal

anatomic structure and function.

WOUND HEALING: It is referred to as phenomenon by which body attempts to restore

the tissue integrity by formation of new structures aimed to replace the defect. The new

structure may more or less match the original structure.

REPAIR : Healing of a wound by tissue that does not fully restore the architecture or

function of the part.

REGENERATION: Reproduction or reconstitution of a lost or injured part.

NEWATTACHMENT: The reunion of connective tissue with a root surface that has been

deprived of its periodontal ligament . This union occurs by the formation of new cementum

with inserting collagen fibers.

REATTACHMENT: The reunion of connective tissue with a root surface on which viable

periodontal tissue was present.

TYPES OF WOUND HEALING

Healing of skin wounds is example of combination of regeneration and repair can be

accomplished in 2 ways

1) Healing by first intension (primary union)

2) Healing by second intension (secondary union)

• Healing by first intention (primary union) is defined as healing of wound which has

following characteristics

• 1)clean and uninfected 2)surgically incised 3)without much loss of cells and tissue

4)edges of the wound approximated by surgical sutures

Sequence

• 1) Initial haemorrahge -occurs immediately after injury. The space between the

approximated surfaces of incised wounds is filled with blood which then clots and seals

against dehydration and infection.

• 2) Acute inflammatory response occurs within 24hours by appearance of

polymorphonuclear neutrophils from margins of incision, by 3rd day they are replaced by

macrophages.

• 3) Epithelial change- basal cells of epidermis from cut margins proliferate and migrate

towards incision space in form of epithelial spurs. Migrated epidermal cells separate the

underlying viable dermis from the overlying necrotic material and clot, forming scab.

• 4)Organization- by 3rd day fibroblast invade wound area ,by 5th day new collagen

fibrils start forming till healing completed ,by 4weeks scar tissue with scanty cellular and

vascular elements ,few inflammatory cells and epithelialised surface is formed.

5) Suture tracks –the mechanism of wound healing is same for suture tracks, since each

suture track is separate wound.

6) When suture is removed around 7th day, epithelial suture tracks is avulsed and remaining

epithelial tissue in track absorbed

Sometimes suture tracks get infected which leads to stitch abscess or epithelial cells persists

in track which leads to implantation or epidermal cysts.

Thus, scar formed in suture wound is neat due to close apposition of margins of wounds

The scab is then cast off; basal cells from margins continue to divide, by 5th day multilayered

new epidermis is formed which is differentiated into superficial and deeper layers.

Healing by secondary intension (secondary union)

Defined has healing of wound having following characteristics

1)open with large tissue defects, at times infected

2)having extensive loss of cells and tissue

3)wound is not approximated by surgical suture but is left open

In secondary union, healing is slow, results in large, at times ugly scar compared to neat scar

and rapid healing of primary.

Sequence

Initial haemorrhage - initially after injury the wound space is filled with blood and fibrin clot

which dries.

Inflammatory phase- initial acute inflammatory response followed by appearance of

macrophages which clear off debris as in primary union.

Epithelial changes- epidermal cells from both margins of wound proliferate and migrate into

wound in the form of epithelial spurs till they meet in middle and re-epithelialise the gap

completely, proliferating epithelial cells do not cover surface fully until granulation tissue

from base has started filling wound space

Feature of secondary wound healing

It is associated with large tissue defect and initially fibrin, necrotic debris and exudate must

be removed. Inflammatory reaction is more intense.

Much larger amount of granulation tissue is formed

Wound contraction is more due to presence of myofibroblast.

GRANULATION TISSUE FORMATION

GRANULATION TISSUE: It is the young vascularised connective tissue with variable

number of inflammatory cells. It appears as granular, pink and soft.

Physiologically it contains: - newly proliferated blood vessels

- Fibroblast like cells and fibroblasts

PHASES OF GRANULATION TISSUE:

1. Phase of inflammation

2 phase of clearance

3 phase of in growth of granulation tissue

A. angiogenesis

B fibrogenesis

ANGIOGENESIS

Initially the proliferating endothelial cells are solid buds but in few hours develop a lumen

and start carrying blood, newly formed blood vessels are leaky so it gives oedematous

appearance of new Angiogenesis-new blood vessels formed by proliferation of endothelial

cells from margins of severed blood vessels.

Granulation tissue, soon they differentiate into muscular arterioles, thin walled venules, and

true capillaries.

FIBROGENESIS

Some of fibroblast has combination of morphologic and functional characteristics of smooth

muscle cells (myofibroblasts).

Newly formed blood vessels are present in amorphous ground substance or matrix, the new

fibroblast originate from fibrocyte and mitotic division of fibroblasts.

Collagen fibers begin to appear on 6th day, as formation and maturation of more and more

collagen takes place, active fibroblasts and new blood vessels decreases resulting in inactive

looking scar known as cicatrisation

COLLAGEN SYNTHESIS

• COLLAGEN-family of protein, provide structural support to multicellular organisms

synthesized and secreted by a complex biochemical mechanism on ribosomes defective

regulation of collagen leads to hypertrophied scar, fibrosis and organ failure.

• Depending on biochemical composition, there are 18 types of collagen fibres are

present.

• Type1, 3, 5 are true fibrillar collagen which form the main portion of the connective

tissue during healing of wounds in scars. Other types are non-fibrillar and amorphous

material seen as component of basement membrane.

Wound contraction

Wound contraction: It is the reduction in size of a wound. Mediated principally by

myofibroblast.

Advantages: Healing of wound is faster

Scar formation is minimal

Mechanism of wound contraction:

1.Dehydration

2. Contraction of collagen

3. Presence of myofibroblasts

Factors inhibiting wound contraction:

1. Corticosteroid administration has inhibitory effect

2. Cytotoxic drugs inhibits wound contraction

3. Radiation interferes wound contraction

Wound strength

• Wound strength depends upon the increase in the amount of collagen present.

Factors influencing wound strength:

1. Direction of the wound

2. Pull of the underlying muscle

3. Previous wounds

FACTORS THAT AFFECT HEALING

In the periodontium, as elsewhere in the body, healing is affected by local and systemic

factors.

Local Factors

Systemic conditions that impair healing may reduce the effectiveness of local periodontal

treatment and should be corrected before, or along with, local procedures. However, local

factors, particularly plaque microorganisms are the most common deterrents to healing

following periodontal treatment.

Healing is also delayed by excessive tissue manipulation during treatment, trauma to the

tissues, the presence of foreign bodies, and repetitive treatment procedures that disrupt the

orderly cellular activity in the

healing process. An adequate blood supply is needed for the increased cellular activity during

healing; if this is impaired or insufficient, areas of necrosis will develop and delay the healing

process.

Healing is improved by debridement (the removal of degenerated and necrotic tissue),

immobilization of the

healing area, and pressure on the wound. The cellular activity in healing entails an increase in

oxygen consumption, but healing of the gingiva is not accelerated by artificially increasing

the oxygen supply beyond the normal requirements.

Systemic Factors

Healing capacity diminishes with age,probably due to atherosclerotic vascular changes,

which are common in aging, and result in reduction in blood circulation. Healing is delayed

in patients with generalized infections and in those with diabetes and other debilitating

diseases. Healing is retarded by insufficient food intake; bodily conditions that interfere with

the use of nutrients; and deficiencies in vitamin C,proteins,and other nutrients. However, the

nutrient requirements of the healing tissues in minor wounds, such as those created by

periodontal surgical procedures, are ordinarily satisfied by a well-balanced diet.

Healing is also affected by hormones. Systemically administered glucocorticoids such as

cortisone hinder repair by depressing the inflammatory reaction or by inhibiting the growth of

fibroblasts, the production of collagen, and the formation of endothelial cells. Systemic

stress,thyroidectomy, testosterone, adrenocorticotropic hormone (ACTH), and large doses of

estrogen suppress the formation of granulation tissue and retard healing.' Progesterone

increases and accelerates the vascularization of immature granulation tissue and appears to

increase the susceptibility of the gingiva to mechanical injury by causing dilation of the

marginal vessels.

GROWTH FACTORS IN WOUND HEALING

Epidermal Growth Factor[EGF]

Platelet Derived Growth Factor[PGDF]

Fibroblast Growth Factor[FGF]

Vascular Endothelial Growth Factor[VEGF]

Transforming Growth Factor[TGF]

FUNCTIONS OF VARIOUS GROWTH FACTORS

Monocyte Chemotaxsis: PDGF, FGF, TGF-

Fibroblast Migration : PDGF, EGF, FGF,TNF, TGF

Fibroblast Proliferation: PDGF, FGF, EGF

Angiogenesis : VEGF, FGF, PDGF

Collagen Synthesis : TGF, PDGF, TNF

Collagen Secretion : TGF, TNF, FGF, EGF

Mitogenic Polypeptides

Lynch etal (1989) used a combination of purified platelet derived growth factor and

recombinant insulin growth factor-1, stimulated periodontal regeneration.One microgram of

each factor is a methylcellulose gel carrier was applied to the root surfaces after full

thickness flaps had been reflected in three dogs with natural periodontitis defects. After a

two week healing interval , conrol sites (7 teeth ) a long junctional epithelial attachment . In

the growth factor treated sites , new cementum and bone formed was lined by osteoblasts.

Lynch et al (1991) conducted a study to evaluate periodontal regeneration in 13 beagle dogs

exhibiting natural periodontitis defects treated with a combination of 3µg of recombinant

platelet derived growth factor –BB and insulin growth factor -1 in a methyl cellulose gel

carrier. Radio labeled growth factor -1 was evaluated in four additional animals to

determine clearance rate . Both factors were cleared rapidly with 96% removed by four days

and essentially all removed by 14 days. The half life approximated four hours for platelet –

derived growth factor and three hours for insulin growth factor.

Epidermal Growth Factor (EGF) and Transforming Growth Factor- (TGF-a).

These two factors belong to the EGF family and share a common receptor. EGF was

discovered by its ability to cause precocious tooth eruption and eyelid opening in newborn

mice. EGF is mitogenic for a

variety of epithelial cells, hepatocytes, and fibroblasts. It is widely distributed in tissue

secretions and fluids, such as sweat, saliva, urine, and intestinal contents. In healing wounds

of the skin, EGF is produced by keratinocytes, macrophages, and other inflammatory cells

that migrate into the area. EGF binds to a receptor (EGFR) with intrinsic tyrosine kinase

activity, triggering the signal transduction events . TGF-a was originally extracted from

sarcoma virus—transformed cells and is involved in epithelial cell proliferation in embryos

and adults and malignant transformation of normal cells to cancer. TGF-a has homology with

EGF,

binds to EGFR, and produces most of the biologic activities of EGF. The "EGF receptor" is

actually a family of membrane tyrosine kinase receptors that respond to EGF, TGF-a, and

other ligands of the EGF family." The main EGFR is referred to as EGFR1, or ERB B1. The

ERB B2 receptor (also known as HER-2/Neu) has received great attention because it is

overexpressed in breast cancers and is a therapeutic target

Platelet-Derived Growth Factor (PDGF). PDGF is a family of several closely related

proteins, each consisting of two chains designated A and B. All three isoforms of PDGF (AA,

AB, and BB) are secreted and are biologically active. Recently, two new isoforms—PDGF-C

and PDGF-D—have been identified. PDGF isoforms exert their effects by binding to two

cell-surface receptors, designated PDGFR alpha and beta, which have different ligand

specificities.

PDGF is stored in platelet a granules and is released on platelet activation. It can also be

produced by a variety of other cells, including activated macrophages, endothelial cells,

smooth muscle cells, and

many tumor cells. PDGF causes migration and proliferation of fibroblasts, smooth muscle

cells, and monocytes, as demonstrated by defects in these functions in mice deficient in either

the A or the B chain of PDGF. It also participates in the activation of hepatic stellate cells in

the initial steps of liver fibrosis

Fibroblast Growth Factor (FGF). This is a family of growth factors containing more than

10 members, of which acidic FGF (aFGF, or FGF-1) and basic FGF (bFGF, or FGF- 2) are

the best characterized. FGF-1 and FGF-2 are made by a variety of cells. Released FGFs

associate with heparin sulfate in the ECM, which can serve as a reservoir for storing inactive

factors. FGFs are recognized by a family of cell-surface receptors that have intrinsic tyrosine

kinase activity. A large number of functions are attributed to FGFs, including the following:

New blood vessel formation (angiogenesis): FGF-2, in particular, has the ability to

induce the steps necessary for new blood vessel formation both in vivo and in vitro .

Wound repair: FGFs participate in macrophage, fibroblast, and endothelial cell

migration in damaged tissues and migration of epithelium to form new epidermis.

Development: FGFs play a role in skeletal muscle development and in lung

maturation. For example, FGF-6 and its receptor induce myoblast proliferation and

suppress myocyte differentiation, providing a supply of proliferating myocytes. FGF-

2 is also thought to be involved in the generation of angioblasts during

embryogenesis. FGF-1 and FGF-2 are involved in the specification of the liver from

endodermal cells.

Hematopoiesis: FGFs have been implicated in the differentiation of specific lineages

of blood cells and development of bone marrow stroma.

TGF-beta and Related Growth Factors. TGF-beta belongs to a family of homologous

polypeptides that includes three TGFR isoforms (TGF-1, TGF- 2, TGF- 3) and factors with

wide ranging functions, such as bone morphogenetic proteins (BMPs), activins, inhibins, and

mullerian inhibiting substance. TGF-131 has the most widespread distribution in mammals

and will be referred to as TGF-/3. It is a homodimeric protein produced by a variety of

different cell types, including platelets, endothelial cells, lymphocytes, and macrophages.

Native TGF-/3s are synthesized as precursor proteins, which are secreted and then

proteolytically cleaved to yield the biologically active growth factor and a second latent

component. Active TGF-/3 binds to two cell surface receptors (types I and II) with

serine/threonine kinase activity and triggers the phosphorvlation of cytoplasmic transcription

factors called Smads. TGF-/3 first binds to a type II receptor, which then forms a complex

with a type I receptor, leading to the phosphorvlation of Smad 2 and 3. Phosphorylated

Smad2 and 3 form heterodimers with Smad4, which enter the nucleus and associate with

other DNA-binding proteins to activate or inhibit gene transcription. TGF-13 has multiple

and often

opposing effects depending on the tissue and the type of injury. Agents that have multiple

effects are called pleiotropic; because of the large diversity of TGF-R effects, it has been said

that TGF-/3 is pleiotropic with a vengeance.

HEALING FOLLOWING SCALING / ROOTPLANING & CURETTAGE

Immediately after curettage, a blood clot fills the pocket area, which is totally or partially

devoid of epithelial lining. Hemorrhage is also present in the tissues with dilated capillaries,

and abundant polymorphonuclear leukocytes appear shortly thereafter on the wound surface.

This is followed by a rapid proliferation of granulation tissue, with a decrease in the number

of small blood

vessels as the tissue matures. Restoration and epithelialization of the sulcus generally require

from 2 to 7 days (Moskow BS 1962 ) and restoration of the junctional epithelium occurs in

animals as early as 5 days after treatment. Immature collagen fibers appear within 21 days.

Several investigators have reported that in monkeys and humans treated by scaling

procedures and curettage, healing results in the formation of a long, thin junctional

epithelium with no new connective tissue attachment. Sometimes this long epithelium is

interrupted by "windows" of connective tissue attachment .

Dubrez etal (1990) reported statistically significant increase in both superficial and deep

average bone densities at six months and one and a half year post scaling and rootplaning.

Cobb etal (1996) concluded from his studies that in patients who adopt proper oral hygiene

measures,

Healing after nonsurgical therapy →complete after about 3-6 months.

The number of sites that bleed on probing will be markedly reduced.

More gingival recession and gain of probing attachment →in intially deeper sites

than more shallow pocket sites.

Initial probing depths of 6-9 mm, the residual probing depth - 4-5mm and the

amount of gingival recession about 2mm.

In sites with pockets of greater than 3mm, a probing attachment loss of 0.5 mm

will occur.

HEALING AFTER SURGICAL GINGIVECTOMY

The initial response is the formation of a protective surface clot; the underlying tissue

becomes acutely inflamed, with some necrosis. The clot is then replaced by granulation

tissue. By 24 hours, there is an increase in new connective tissue cells, mainly angioblasts,

just beneath the surface layer of inflammation and necrosis; by the third day, numerous

young fibroblasts are located in the area . The highly vascular granulation tissue grows

coronally, creating a new free gingival margin and sulcus.

Capillaries derived from blood vessels of the periodontal ligament migrate into the

granulation tissue,

and within 2 weeks they connect with gingival vessels . After 12 to 24 hours, epithelial cells

at the margins of the wound start to migrate over the granulation tissue, separating it from the

contaminated surface layer of the clot. Epithelial activity at the margins reaches a peak in 24

to 36 hours; the new epithelial cells arise from the basal and deeper spinous layers of the

wound edge epithelium and migrate over the wound over a fibrin layer that is later resorbed

and replaced by a connective tissue bed. The epithelial cells advance by a tumbling action,

with the cells becoming fixed to the substrate by hemidesmosomes and a new basement

lamina.

Surface epithelization is generally complete after 5 to 14 days. During the first 4 weeks after

gingivectomy, keratinisation is less than it was prior to surgery. Complete epithelial repair

takes about 1 month. Vasodilation and vascularity begin to decrease after the fourth day of

healing and appear to be almost normal by the 16th day .Complete repair of the connective

tissue takes about 7 weeks.

The flow of gingival fluid in humans is initially increased after gingivectomy and diminishes

as healing

progresses. Maximal flow is reached after 1 week, coinciding with the time of maximal

inflammation( Arnold Et al 1966)

Loss of attachment has been reported after gingivectomy- Walt 1976.

There is a slight reduction in the width of attached gingiva- Glickman 1966.

An increase in tooth mobility immediately after gingivectomy has been reported with

patterns returning to presurgical levels after healing - Burch etal 1960

Although the tissue changes that occur in post gingivectomy healing are the same in all

individuals, the

time required for complete healing varies considerably, depending on the area of the cut

surface and interference from local irritation and infection. In patients with physiologic

gingival melanosis, the pigmentation is diminished in the healed gingiva.

HEALING AFTER FLAP SURGERY

Immediately after suturing (0 to 24 hours), a connection between the flap and the tooth or

bone surface is established by a blood clot, which consists of a fibrin reticulum with many

polymorphonuclear leukocytes, erythrocytes, debris of injured cells, and capillaries at the

edge of the wound. A bacteria and an exudate or transudate also result from tissue injury.

One to 3 days after flap surgery, the space between the flap and the tooth or bone is thinner,

and epithelial cells migrate over the border of the flap, usually contacting the tooth at this

time. When the flap is closely adapted to the alveolar process, there is only a minimal

inflammatory response .

One week after surgery, an epithelial attachment to the root has been established by means of

hemidesmosomes and a basal lamina. The blood clot is replaced by granulation tissue derived

from the gingival connective tissue, the bone marrow, and the periodontal ligament.

Two weeks after surgery, collagen fibers begin to appear parallel to the tooth surface.' Union

of the flap to the tooth is still weak, owing to the presence of immature collagen fibers,

although the clinical aspect may be almost normal.

One month after surgery, a fully epithelialized gingival crevice with a well-defined epithelial

attachment is present. There is a beginning functional arrangement of the supracrestal fibers.

Full-thickness flaps, which denude the bone, result in a superficial bone necrosis at 1 to 3

days; osteoclastic resorption follows and reaches a peak at 4 to 6 days, declining thereafter."

This results in a loss of bone of about 1 mm ; the bone loss is greater if the bone is thin

Osteoplasty (thinning of the buccal bone) using diamond burs, included as part of the surgical

technique, results in areas of bone necrosis with reduction in bone height, which is later

remodeled by new bone formation.

Therefore the final shape of the crest is determined more by osseous remodeling than by

surgical reshaping. This may not be the case when osseous remodeling does not include

excessive thinning of the radicular bone. Bone repair reaches its peak at 3 to 4 weeks.

HEALING AFTER FREE SOFT TISSUE GRAFTS

Initial /plasmic circulation phase (0-3day)

A thin blood clot forms between the transplant tissue and periosteal connective tissue bed

.Marked inflammation but no vascularisation prior to the 3rd postoperative day. Tissue

survives via diffusion from recipient site, termed plasmic or plasmatic circulation. Graft

appears edematous until then. It must remain immobile with a thin underlying clot.

Should be sufficiently smooth to allow for close adaptation.Digital pressure should be

applied for several minutes . In early period , the epithelium desquamates to be restored

once the blood supply is reestablished. Third day , there begins a gradual return of

circulation and a reddish color.

Revascularisation(4-11 days)

After 4-5 days→ blood vessels of the recipient site join the grafted tissue , reestablishing

circulation.

Blood clot is gradually resorbed and replaced by connective tissue that united the

graft and recipient area. Adequate blood supply is achieved by about the 8th day and

fibrous attachment by 10th day.

Early on this phase , the tissue is red and becomes gradually more pink as the blood

supply is restored. The graft reepithelises by proliferation of the epithelium remnants in

its retepegs and form contiguous epithelized tissues. There is formation of long

junctional epithelium

Tissue maturation / organic union phase (12-42 days)

Exuberantly renewed vascular plexus is reduced to normal within about 14 days. There

is replacement and maturation of epithelium and keratinisation. Pink color is restored

during this phase. Donor site heals by secondary intention with epithelisation coming

from the wound margins over the remaining connective tissue substrate. Movement of

epithelial cells- 0.5-1mm of lateral progress per day. Healing time is related to the

surface area of the wound.

Creeping attachment

Root coverage seen in early phase of healing was earlier termed Budging and later

creeping attachment was seen. The term was described by Goldman etal (1964) as the

post operative migration of the gingival margin in a coronal direction over a previously

denuded root. This migration continue for a long time until a constant margin level is

reached.

Clinical importance is the few mm increase in root coverage.

Matter and Cimmasoni (1980) suggested that it is more likely to occur in

Narrow defects

Younger patients

Single root recession

Original tooth position

HEALING IN THICK VS THIN GRAFT

Bridging is the Phenomenon of graft survival over an avascular root surface .

Sullivan and Atkins- The two point collateral circulation present toward the coronal portion

of a free graft over avascular root surface was insufficient to maintain tissue viability

particularly in deep wide recessions

Graft thickness would determine its behaviour during healing and its final

character .Thinner grafts (0.5-0.7mm) enhanced survival

Thick graft with a thicker lamina propria had greater primary contraction causing blood

vessels to collapse , retarding revascularization and reducing the likelihood of bridging.

However once healed thicker grafts show superior resistance to frictional stress and are

recommended for areas with high susceptibility for gingival recession.

Healing after connective tissue graft (Caffesse etal2001)

At 7days- Oral epithelium of flap there is absence of retepegs. Superficial location of the

interface between the graft and flap showed deeper projection of the retepeg. .

In the Clot , there were immature fibres that formed a network in which the blood cells and

inflammatory cells get trapped.

At 14 days- JE is not yet formed .Granulation tissue between flap and the graft is more

organized and more compact than in one week specimens. More blood vessels are seen.

At 28 days there is increase in the thickness of the sulcular epithelium and junctional

epithelium .Retepegs of oral epithelium is short and stumpy. Demarcation zone between the

graft and the periosteum and between the graft and the overlying flap could not be

delineated .Connective tissue consisted well organized bundle of thicker fibres.

At 60 days - Epithelium regained its normal shape, thickness and appearance .Connective

tissue is well organized and dense mature collagen fibres were present.

COMPLICATIONS IN WOUND HEALING

Retarded epithelisation

Failure of epithelial keratinisation.

Flap displacement and evulsion.

Bone exposure.

Periodontal abscesses, pyogenic granuloma, giant cell reparative granuloma.

Increased Tooth mobility

CONCLUSION

The healing of wound is one of the most interesting of the many phenonena that

characterise the living organism. Only profound understanding of biological and clinical

variables affecting the outcome of periodontal treatment procedures will allow

clinicians to manipulate biological and clinical factors effectively in order to optimize

the clinical result and increase the predicatbility of therapy.

Wound healing is a complex process especially when compomised by local and systemic

factors and is associated with different forms of collagen and different components of

the complement.

The basic healing processes are the same after all forms of periodontal therapy.

Regeneration, repair and new attachment are aspects of periodontal healing that have a

special bearing on the results obtainable by the treatment. The effectiveness of

periodontal therapy is made possible by the remarkable capacity of periodontal tissues.

REFERENCES

Carranza,Clinical Periodontology. 10th edition.

Clinical periodontology jan Lindhe-5th edition.

Ulf m, Wikesjo O, Rolf E Nilveus. Significance of early healing events on

periodontal repair. A review. J Periodontol 1992,

General Pathology-Robins

Bartold, Sampath Narayan, Biology of periodontal connective tissues.

Basic considerations of wound healing.Periodontology 2000 vol 19

Boretti et al.Short tem effects of phase I therapy on crevicular cell population. J

Periodontol 1995;235-240

Cobb C, Non surgical periodontal therapy.Ann Periodontol 1996.