Inflammation
Acute
Marked by
1) changes in vessels a.
flow and caliber b.
Permeability
2) Cellular events a.
Accumulation of neutrophils and macrophages i.
Margination – Adhesion – Chemotaxis – Phagocytosis
3) Chemical Mediators a.
From cells i.
Preformed – histamine, serotonin ii.
Synthesised
1.
PAF
2.
cytokines, growth factors

3.
arachidonic acid derivatives a.
cyclooxygenase pathway – Prostaglandins b.
lipoxygenase pathway – leukotrienes b.
from plasma i.
Complement system (classic, alternate) ii.
Kinin system iii.
Clotting/fibrinolytic system c.
From injury i.
O2 radicals
Chronic
Marked by
1) infiltration by macrophages, lymphocytes and plasma cells
2) proliferation of fibroblasts and small blood vessels
3) increased connective tissue – fibrosis
4) tissue destruction
Granulation Tissue
Formed during the proliferative phase of inflammation
Fragile tissue composed of matrix of fibrin, fibronectin, GAGs with proliferating endothelial cells and fibroblasts mixed with a population of macrophages and neutrophils.
The ability to release these molecules gives rise to the concept that the macrophage plays a pivotal role in regulation of healing (Clark, 1996). This is supported by the demonstration that depletion of macrophages from the wound site inhibits healing (Liebovich & Ross, 1975), that defective healing in aged mice

can be restored by transfer of macrophages from young mice (Danon et al, 1989) and that the proliferation of fibroblasts and endothelial cells during granulation tissue formation follows on from the increase in macrophages during the late inflammatory phase of healing.
Cytokines and growth factors are polypeptide molecules that regulate migration , proliferation , differentiation and metabolism of cells. They may act in:
1) paracrine manner on cells adjacent to the secreting cell
2) autocrine factors on the secreting cell and
3) bound to carrier proteins as endocrine factors.
A diverse range of these factors have been identified in wound interstitial fluid and a number have been identified as potentially playing a key role in regulating healing. Platelet derived growth factor (PDGF) and Transforming Growth Factor
(TGF) are released by platelets and are important in initiating healing. The inflammatory phase of healing is modulated by cytokines such as Tumour
Necrosis Factor alpha (TNFa), Interleukin-1 (IL-1), Interleukin-4 (IL-4) and the peptide chemokine Interleukin-8 (IL-8). The sequential phases of granulation tissue formation, re-epithelialisation and extracellular matrix formation are regulated by Fibroblast Growth Factors, Transforming Growth Factors and
Epidermal Growth Factor amongst many others.
PDGF is a family of three isoforms, composed of dimers of the PDGF-

or
PDGF-

chains, which have overlapping but distinct biological properties generated by interaction with two types of receptor. It is produced by platelets, macrophages, endothelial cells and keratinocytes. Release of PDGF by platelets is important in initiating healing . It stimulates chemotaxis of fibroblasts, neutrophils and macrophages. Once these cells are attracted to the wound site
PDGF can then activate macrophages and induce proliferation of fibroblasts.
Additionally it can stimulate the production of the extracellular matrix components fibronectin and hyaluronan although not as effectively as other factors such as
TGF

.

Recombinant PDGF has been evaluated in several clinical trials to treat nonhealing chronic wounds and has been demonstrated to be of benefit in treatment of wounds where healing is impaired by diabetes (Brown and Breeden,1994).
The three isoforms of TGF

(

1,

2 and

3) have a broad range of activity within healing. TGFb1 is the most abundant in all tissues and is the form found in platelets.
All cells involved in healing can produce and/or respond to TGF

.
Release of TGF

by platelets at the same time as PDGF is important in initiating healing as, at low concentrations, it is chemotactic for monocytes, lymphocytes, and fibroblasts. Its role in angiogenesis is controversial as in some experimental systems it stimulates endothelial cell proliferation and tubule formation whilst in others it is inhibitory. Its actions may therefore be contextual and concentration dependent.
TGF

plays a central role in regulating maturation and strength of wounds .
It regulates many matrix proteins including collagen, proteoglycans, fibronectin, matrix degrading proteases and their inhibitors. Topical application of TGF
 increases the strength of experimental incisional wounds (Mustoe et al 1987).
Manipulation of the TGF

isoforms during healing can modify scarring. Antibody neutralisation of TGF

1 and

2 or increasing TGF

3 concentrations by exogenous application decreases post surgical scarring (Shah et al 1995).
Although at least ten fibroblast growth factors (FGFs) family members are known
FGF-1 (acidic FGF or

FGF) and FGF-2 (basic FGF or bFGF) are most widely characterised with respect to wound healing. They are weakly soluble with a strong affinity for heparan sulphate leading to them being bound in the wound extracellular matrix. FGF-2 has been detected at the wound site early in healing and its rapid appearance after injury suggests that pre-existing tissue FGF-2 may be important in healing rather than that synthesised de novo by inflammatory macrophages. It stimulates angiogenesis leading to accelerated reepithelialisation after experimental application(Mustoe TA et al, 1991).
Epidermal growth factor (EGF) is a small molecule which exhibits homology with regions of the TGF

molecule. It is produced by macrophages and epidermal cells with the keratinocyte and fibroblast as targets. Its primary role is to stimulate keratinocytes to migrate across the wound provisional matrix and induce epidermal regeneration(Brown et al 1991)

The term cytokine generally refers to molecules such as the Interleukins which have initially been investigated for their role in regulation of the immune response. Because of the inflammatory response that is associated with wounded tissue they also have a potential role to play in regulation of healing either directly by their effect on the structural cells in wounded tissue such as fibroblasts, endothelial cells or keratinocytes, or indirectly by their modulation of growth factor production by macrophages.
IL-1 and TNF

are both pro-inflammatory cytokines that will induce expression of adhesion molecules such as ICAM-1 and E-Selectin by endothelial cells allowing leukocytes to adhere to the lumen of capilleries and extravasate into the wound site. These cytokines activate macrophages and initiate production of growth factors required for healing and more pro-inflammatory mediators to prolong the inflammatory response. Overproduction of TNF

may lead to a persisting chronic inflammatory response that is involved in the pathogenesis of chronic wounds.
However there is a requirement for TNFa to allow normal healing (Lee et al 1999) and induction of a transient TNF

response may re-initiate healing in non-healing chronic wounds (Moore 1999).
In order for monocytes to differentiate into activated macrophages they need to pass through a priming stage of differentiation. During this stage they are programmed for responsiveness to subsequent stimuli by exposure to the cytokine microenvironment. For example interferon-g will give a positive priming signal whilst exposure to interleukin-4 acts to down regulate the inflammatory response by inhibition of priming.
Many other factors are likely to be involved in the regulation of healing including
Vascular Endothelial Cell Growth Factor (VEGF), Insulin Like Growth Factor
(IGF-1), Granulocyte Monocyte Colony Stimulating Factor (GM-CSF).
Collagen is the major protein component of the extracellular matrix (ECM) of skin and composes 60-80% of its dry weight. Wound ECM is composed of a mixture of collagen and elastin fibrils interspersed with glycosaminoglycans, long unbranched polysaccharides and proteoglycans, glycosaminoglycans combined with protein. Whilst the ECM fulfills an important structural role it also plays a bioregulatory role in modifying the behaviour of cells that come into contact with it and also by acting as a reservoir of bound growth factors and enzymes.

Collagens are composed of 3 alpha chains which have a common repeating Gly-
X-Y motif that allows folding into a triple helix. Thirty two distinct alpha chains are known allowing the existence of at least nineteen different collagen types in vertebrates. Of these only 7 are found in significant quantities in skin. These are
Collagens I, III, V, XII and XIV which form structural fibrils, Collagen VI which forms microfilaments, Collagen V which forms reticular fibres and Collagen VII which forms fibrils anchoring the epidermis to the dermis.
Collagen is synthesised primarily by wound fibroblasts. It is released at the ribosome as a three chain molecule which then undergoes post translational modification to form procollagen. These modifications include prolyl and lysyl hydroxylation and glucosyslation and galactosylation of lysyl and hydroxylysyl residues (Kivirikko & Myllyla, 1985). The trimeric molecule is then secreted into the extracellular space where mature collagen is formed by the proteolytic cleavage of procollagen peptides. The collagen molecules are then able to associate into fibrils which are stabilized by intermolecular cross link formation.
The cross linkages acquire greater stability as the healing process continues.
Collagen synthesis is regulated by the co-ordinated actions of a number of cytokines. TGF-

1 mRNA co-expresses with Collagen

1 mRNA early in healing and IL-4 and low concentrations of IL-1 stimulate production of Collagen I by fibroblasts in vitro. Counter regulation has been demonstrated by high concentrations of IL-1, Interferon-g and TNF

. (Eckes et al,1996).
The other major ECM components are the proteoglycans. These are proteincarbohydrate complexes characterised by their glycosaminoglycan (GAG) component. GAGs are highly charged sulphated and carboxylated polyanionic linear polysaccharides. Those most commonly present within the ECM are
Hyaluronan, Chondroitin Sulphate, Dermatan Sulphate, Heparan Sulphate and
Keratan Sulphate.
Hyaluronan (HA), previously known as hyaluronic acid, consists of alternating glucuronic acid and N-acetylglucosamine units. Each repeating disaccharide unit has one carboxyl, four hydroxyl and an acetamido group. It differs from the other major GAGs in that it does not possess any sulphate groups and is not covalently linked to proteins to form proteoglycans although it can non-covalently bind proteins and cell surface receptors such as the CD44 molecule. It is synthesised by fibroblasts and is a major component of wound ECM being highly hydrated and conferring viscosity to tissues and fluids.
HA is a component of normal skin and is present throughout the entire healing process. It has the potential to modulate cell function by its physicochemical properties, by acting as a hygroscopic osmotic buffer, its viscoelasticity, its chemical properties such as free radical scavenging, anti-oxidant effects and ability to exclude enzymes from the local cellular environment. Additionally it may interact directly with cells via the RHAMM receptor (Receptor for HA Mediated

Motility), the CD44 receptor and the ICAM-1 (Inter-Cellular Adhesion Molecule -
1) receptor. Receptor interaction may be via a ligand receptor type interaction
(CD44) or via a receptor blockade (ICAM-1).
HA has the potential to interact in each phase of healing. During the inflammatory phase of healing wound tissue is rich in HA. It may play multiple roles promoting inflammation early in healing by enhancing leucocyte infiltration and also moderating the inflammatory response as healing progresses towards granulation tissue formation.
Granulation tissue is also rich in HA and here its role includes facilitation of cell migration (fibroblasts and endothelial cells), into the provisional matrix, Although
HA has not been demonstrated to be mitogenic its oligosaccharide derivatives do stimulate endothelial cell proliferation (West & Kumar, 1989).
HA is present in high concentrations in the basal layer of the epidermis in normal skin and co-localises with the CD44 receptor expressed by keratinocytes migrating over the provisional matrix of wound granulation tissue. Suppression of
CD44 expression and consequential decreased HA binding results in defective inflammatory responses, decreased skin elasticity and impaired healing.
In contrast to wounds in the adult, foetal wounding is characterised by a lack of fibrotic scarring. The HA content of foetal wounds remains high for longer periods than in adult wounds. Whilst it is attractive to confer a causal relationship between these two observations many other differences exist between the foetal and adult wounds. For instance the foetal wound is a sterile environment with a relatively lower capacity for generation of the early inflammatory phase of healing. However applied HA has been demonstrated to induce scarless healing of adult tympanic membranes.
Collagen is constantly being degraded and re-synthesised even in normal intact skin. Following injury its rate of synthesis increases dramatically along with an increase in degradation. Collagen synthesis decreases to normal levels by day
21 after wounding. Remodelling of the collagen fibres by degradation and resynthesis allows the wound to gain strength by re-orientation of collagen fibres.
The resulting scar is less cellular than normal skin and never achieves the same tensile strength as uninjured skin.
Collagen degradation requires specific enzymes known as collagenases. These are members of the matrix metalloproteinase (MMP) family. MMPs are produced by a number of cell types including, macrophages, fibroblasts and keratinocytes.
Their production is inducible and regulated by cytokines, growth factors, hormones and contact with ECM components. Pro-inflammatory cytokines such

as TNF

and IL-1 appear to be major inductive factors whilst TGF-

inhibits procollagenase production and induces synthesis of specific inhibitors of MMPs known as TIMPs (Tissue inhibitors of metalloproteinases).
Remodelling can continue for up to two years after injury as the healed wound becomes covered with mature tissue. The relative weakness of the scar compared to normal skin is a consequence of the collagen fibre bundle orientation and abnormal molecular cross linking. The fibres in normal skin are relatively randomly ordered whilst in scar tissue more of the fibres run in parallel.
As well as poor cosmesis scar tissue tends to contract abnormally so that normal function can be lost where there are large areas of scarring such as following burn wounds.