repair of skin wounds by fibrosis

8/6/2019 Repair of Skin Wounds by Fibrosis

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REPAIR OF SKIN WOUNDS

BY FIBROSIS


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Functions of the skin

Thermoregulation (insulation & evaporation)

Sensory transduction - Detects touch,

pressure, pain and temperature

Protects underlying tissues and organs by acting

as mechanical barrieragainst desiccation,

invasion by microbes, environmental insults

such as UV irradiation, mechanical trauma,chemical or thermal burns

Excretes salts, water and organic wastes

(through the sweat glands)

Synthesis vitamin D3


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Dermis no regeneration

Repair by fibrosis

Pig skin = human skin model Most studies rodents or rabbit

breeding, handling and maintenance

Wound healing studies difficult tocompare differences in model, strain,

sex, age, location and size of wound


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STRUCTURE OF THE SKIN


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Structure of the Adult Mammalian skin

Two main layers epidermis & dermis EPIDERMIS

stratified squamous epithelium

consisting primarily ofkeratinocytes from various

stages of differentiation, from mitotically active basalcells (stratum basale-deepest layer) to the heavily

keratinized superficial cells (stratum corneum) -

sloughed off

Keratinocytes impermeable sheet tight junctions,desmosomes

Stem cells in stratum basale - constantly divide to

self renew and give rise to differentiated

keratinocytes that migrates upwards to replace the

cells of the stratum corneum as they slough off


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THE 5 SUBLAYERS OF EPIDERMIS


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EPIDERMIS

Non epithelial celltypes :

Melanocytes-colour

Langerhans cells -APCs

Merkel cells -mechanoreceptors


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The ECM of the stratum basale contains

hyaluronic acid (HA) for which the basal cells

express the CD44 receptor.


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DERMIS

located beneath the epidermis two layers of fibroblasts which are embedded in

ECM ;

The Papillary layer- next to the basal layer of the

epidermis papillae

DeeperReticular layer

contains most of the skins specialized cells and

structures, including: Blood & Lymph vessels, Hairfollicles, Sweat & Sebaceous glands, Nerve

endings, Collagen & Elastin


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The Papillary Layer

thrown into folds of highly vascularized

loose connective tissue

pervaded by a capillary network -nourishment to the epidermis, and acts as

a heat exchanger

ECM thin collagen and elastic fibers;

mast cells; tissue macrophages; fat cells


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The Reticular Layer

thicker than the papillary layer

characterized by an ECM containing a network

ofcoarse collagen & elastin fibers

larger blood vessels and fewer capillaries

The reticular layer rests on a superficial fascia

or hypodermis (not part of the skin)

Bundles of collagen fibers extending fromreticular layer anchor it onto the hypodermis


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Proteins of the Dermal ECM

three classes Proteoglycans, Fibrous Proteins,Adhesive Proteins


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PROTEOGLYCANS

proteins linked to sulfated

GAGs

Dermatan sulfate, heparan

sulfate & chondroitin sulfate -

prominent in dermal ECM

Significant PGs in dermalECM are the large PG

versican, and the small PG

decorin

Multiple PGs linked to HA Binds water - - resisting

compressive forces and space

for cell migration in injured skin


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FIBROUS PROTEINS

Major Fibrous Proteins in the Dermal ECM

Collagens - give tensile strength

type I (80%) and type III major

Type VI - forms a highly branched network offilaments surrounding the type I collage fibrils

type IV - part of basement membrane of blood

vessels

type VIII - located around hair follicles, andsmall blood vessels

Elastins give resiliency - allowing the skin to be

stretched and then assume its original shape


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The Dermal Adhesive Proteins

Prominent - Fibronectin (Fn), Vitronectin (Vn),

and Tenascin-C (Tn-C)

Fibronectin and Vitronectin serve as a

substrate to which cells can adhere when they

are either migrating or stationary.

Tenascin-C is an antiadhesive protein - with

Fibronectin, helps control the degree of cellularadhesion to the ECM substrate


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Integrins

Proteins of dermal ECM aa recognitionsequences receptor binding

Major cell receptor forECM molecules such

as collagen I, III, and Fibronectin low affinity, heterodimeric linker proteins

consisting of two non covalently associatedtransmembrane glycoprotein subunits and

Functions: adhesion of cells to ECM

migration of cells on ECM

maintenance and modulation of gene expression by

the transmission of signals to the nucleus


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Epidermal Growth Factor Receptor- Tenascin-C

and type I collagen have EGF repeat domains -

bind to EGFR

Other recognition domains protein binding

organization of ECM

Basement membrane synthesized byepidermis connects with papillary layer

Composed of laminin in lamina lucida

Type IV collagen, entactin and perlecan lamina

densa Epidermal cells lamina lucida hemidesmosomes

and integrins

Lamina densa- papillary layer type VII collagen


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The dermal ECM - reservoir for GFs - binding &

releasing them upon injury GFs are signaling molecules that stimulate or

inhibit proliferation, migration and differentiation,

depending upon the cell type

Most GFs - RTK pathways - initiate intracellularphosphorylation cascades by other kinases,

resulting in activation of transcription factors that

up-or down-regulate gene activity

TGF-beta Smad pathway

ECM, GFs, CAM and GF receptors imp for

wound healing


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The Effect of Wound Type & Extent on

Dermal Repair

Injury to the epidermis layer alone - regeneration

without scar formation

wounds that penetrate the dermis - repair by

fibrosis Wounds made by Shallow surgical incisions

(superficial) - simple fibroblast proliferation with

minimal scar formation - little wound space to be

filled in

Deep Incisions, excisional wounds and burns -

wound edges - far apart destruction of

substantial amounts of tissue - repair by the

formation of extensive scar tissue


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Phases of Repair in Excisional Wounds

3 tightly integrated phases - occupy variable timeframes, depending on size of wound:

Hemostasis

Inflammation

Structural Repair

Nine cell types - major roles in the epidermal and

dermal repair process:

Platelets, neutrophils, macrophages, T-cells, mastcells, injured axons of sensory and sympathetic

post-ganglion nerves - hemostasis and inflammation

Epidermal cells, dermal fibroblasts and endothelial

cells - means for structural repair


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Phase I Hemostasis & Clot

Formation

Wound - blood vessels, epidermal and

connective tissue cells & surrounding ECM

First response to wounding HEMOSTASIS -

minutes to stop bleeding and seal off the wound

by 3 mechanisms:

a) Formation of Platelet Clumps

b) Vasoconstrictionc) Formation of Fibrin Clot


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a) Formation ofPlatelet Clumps

The cells in the wall of blood vessel at the site of injury -

release ADP - clumping of platelets to the injury site

Immediately upon an injury, blood flood into thewounded area

Degranulation of clumped platelets upon contact with

exposed collagen in the walls of the injured blood vessels

releasing more ADP (further attracts more platelets),Arachidonic acid, Fibrinogen, Fibronectin,

Thrombospondin, and von Willebrand Factor VIII

AA - converted to thromboxane A2 through COX pathway


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b) Vasoconstriction

Thromboxane A2 and serotonin from injured

nerve axons vasoconstrictors - restrict blood

flow into the wound by constricting the blood

vessels The injured nerves - substance P , a

neuropeptide - mast cells degranulation in the

dermis - releasing histamine increase in the

permeability of vessel walls -allowing furtherleakage of plasma into the wound


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c) Formation of Fibrin Clot

Blood plasma in the wound space contains coagulationfactors - induce clot formation

Hagemann Factor VII, a plasma protein initiates a

cascade of reactions involving about 12 clotting factors (I-

XII) and requireC

a

2+

as an essential cofactor. Tissue factor - produced at the end of cascade and

converts prothrombin to active enzyme thrombin

Thrombin catalyzes the conversion of plasma fibrinogen

to fibrin, the major structural protein of clot

Prothrombin Thrombin

Tissue factor

Fibrinogen Fibrininsoluble clot


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Fibrin molecules -e organized into fibers that

intertwine to form a meshwork - traps RBCs,

WBCs and platelets Meshwork also contains plasma Fibronectin,

Vitronectin as well as Thrombospondin from

degranulated platelets and collagen types I,

III, IV (probably synthesized by monocytes)

Blood Clot Formation (blood cells, platelets, fibrin clot) (SEM x10,980)


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ADP


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Contraction of provisional matrix and

exudation of serum thrombosthenin

Dehydration of clot scab prevents fluidloss

Invasion by cells of immune system


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Phase 2: Inflammation

Within a day after injury, chemotactic factors released during

clot formation

Increase in permeability of surrounding capillaries - attracts

neutrophils, monocytes and T-lymphocytes - crawl out

between endothelial cells into the provisional fibrin matrix

The leukocytes insert themselves into spaces between

endothelial cells via binding of Platelet Endothelial Cell Adhesion

Molecule (PECAM) on the surface of the leukocytes to PECAM

on extrusions of endothelial cell surface.

Neutrophils and monocytes migrate into the clot

simultaneously; neutrophils in greater numbers - use fibronectin

in the clot as an adhesive substrate and express the appropriate

integrin receptor to bind to fibronectin


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After entering the wound and adhering to Fibronectin, the

monocytes differentiate into macrophages

Within the clot, macrophages secrete TGF-, PDGF and other

chemoattractants such as leukotriene B4, monocyte-

chemoattractant proteins 1,2,3 (MCPs), macrophageinflammatory protein 1 and , and the chemotactic protein

CAP37 attract neutrophils and macrophages

Neutrophil influx diminishes within 3-4 days after injury. T-cell

lymphocyte peaks by the end of the first week or later. T cells

particularly the Th1 subset may play a role in regulating

macrophage-induced activities.


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Phase 2: Inflammation

Lasts for 5-7d

Within a day - Chemotactic factors - released

during platelet degranulation and clot formation

including fibrinopeptides (fibrinogen cleavageby thrombin) , fibrin degradation products

produced by the action ofplasmin,

complement fragment C5a, collagen & elastin

fragments

The most important chemoattractants for the

inflammatory phase are TGF- and PDGF

supplied by degranulating platelets.


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TGF- - attracts monocytes

PDGF - attracts neutrophils and monocytes

tPA plasminogen to plasmin

Plasmin activates MMPs and also acts

directly on fibrin


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Important tasks of inflammatory

neutrophils and macrophages

kill bacteria through oxygen-dependent mechanisms that

generate H2O2 and HOCl

Neutrophils also produce bactericidal peptides and

proteins, including the enzyme cathepsin G Neutrophils and macrophages also play a central role in

degrading collagen within the wound - production of

MMPs - MMP-1 and MMP-8

MMP-1 degrades type I and III collagens & MMP-8

degrades type I collagen.

The neutrophils and macrophages clean up the wound

site by phagocytizing dead bacteria, as well as cellular

and molecular debris.


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Neutrophils accelerate keratinocyte

differentiation slowing proilferation andmigration

Continual influx of neutrophils andmacrophages to replace the dead ones

may lead to chronic tissue injury, necrosis andexcessive scarring

Limit to their migration and secretoryactivities and also their removal

Neutrophils apoptosis after few hrs in thewound engulfed by macrophages


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Phase 3 : Structural Repair

Several sub phases:

Re-epithelialization

Formation of Granulation Tissue

Remodelling of Granulation tissue into scar


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Phase 3: Structural Repair

a) Re-epithelializationWithin a few hours after injury - cells in stratum basalelayerof the epidermis at the edge of wound loosen their

attachments to one another and begin migrating as a

sheet through the fibrin clot to cover the wound surface

The migrating cell sheet just expands (does not divide) -

basal cells just interior to the wound edge divide and

feed progeny into the migrating sheet

After the migrating epidermis has expanded to cover the

wound surface, its cells divide vertically to thicken the

epidermis and synthesize a new basement membrane

Excessive damage no regeneration of hair and sweat glands


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b) Development of Granulation Tissue

Within two days after injury, well before the inflammatory

phase is over, fibroblasts begin migrating from the

surrounding dermis into the clot.

Initiated by GFs secreted by macrophages, primarily

PDGF and TGF- - stimulate fibroblast migration and

proliferation

Replacement of fibrin clot by capillary-rich fibroblastic tissue

Simultaneously , new capillaries regenerate into the

nascent fibroblastic tissue, which is now called

granulation tissue because the capillaries give it a

reddish, granular appearance


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Macrophages, fibroblast and capillaries invade the wound

space as a unit biologically interdependent during tissue

repair

The fibroblasts synthesize a new transitional matrix to

replace the fibrin matrix, and the new capillaries carry

oxygen and nutrients to the injury site, to sustain the

metabolism required for these cellular process.


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Source of fibroblasts for structural repair: Resident dermal fibroblast

present in the deep layers of reticular dermis

and hypodermisSynthesize collagen type I

Fibroblast differentiating from circulating

mesenchymal stem cells from bon marrowthat enter the wound from the vasculature.

Synthesize collagen type I and III

Fibroblast Migration and Proliferation


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Heterogeneity in fibroblast population

Sub population of skin fibroblasts show:

1) Variation in morphology.

2) Variation in the amount of collagen

expressed per cell.

3) Variation in their response to cytokines

derived from macrophages

Papillary layer fibroblasts higher metabolic

rate and proliferative activity and cell densityat confluence

Heterogeneity in fibroblasts from healing intra-

oral wounds and fetal dermis


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The movement of fibroblasts into thefibrin matrix of the wound - mediated by

their integrin receptors Fn and HA major components of early

granulation tissue matrix

HA-PG complexes bind water expandEC space fibroblast migration

The fibroblasts express CD 44 receptor -mediates cell attachment to and

locomotion on HA substrate andreceptor for hyaloronan-mediatedmotility (RHAMM) which mediates celllocomotion in response to soluble HA


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Fibronectin

acts as asubstrate for

the migration

of fibroblasts

and vascular

endothelial

cells into the

wound

b bl l f d


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Fibroblasts proliferation, migration and ECM

synthesis in vitro - stimulated by macrophages

through GFs during inflammatory phase TGF

and PDGF

Growth factors:

TGF- (Transforming growth factor) PDGF (Platelet-derived GF) major GF renders

fibroblasts competent to leave G0 and enter G1

Receptor on dermal fibroblasts

FGF-2 (Fibroblast GF)

EGF (Epidermal GF)

IGF-1 (Insulin-like GF)


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Cytokines:

PDGF produced by macrophages

IL-1 (Interlukin) by macrophages

stimulates fibroblasts to produce PDGF

IFN- inhibits fibroblast entry into G1

TGF- & TNF- (Tumor necrosis factor)

stimulate fibroblast proliferation


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As the number offibroblasts invading the woundspace increases and the number of neutrophils

decreases, the fibrin provisional matrix isdegraded into soluble fragments through theconversion ofplasminogen toplasmin by tPA(tissue plasminogen activator) secreted by

endothelial cells of regenerative capillaries, andMMPs (Matrix Metallo Proteinases) secreted byfibroblasts.

When the fibrin degrades, tPA is inhibited and theexpression ofplasminogen activatorinhibitor(PAI) is stimulated, thus reducing theconversion of plasminogen to plasmin.


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ECM Synthesis By Fibroblasts

As fibroblasts invade the wound space, theyreplace the provisional fibrin matrix with an ECM

consisting ofFn, HA, sulfated PGs and type I and III

collagens.

HA predominates over Fibronectin in early

granulation tissue, opening up migration space for

fibroblasts.

Type IIIis the major collagen synthesized at this

time organized in reticular pattern


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Dermal repair then show visible signs of fibrotic

rather than a regenerative pathway.

HA synthesis is replaced by synthesis ofchondroitin

sulfate and dermatan sulfate-PGs.

The fibroblasts synthesize predominantly Type I

collagen and more collagen is synthesized than in

uninjured skin.

Shift in pattern of ECM synthesis PDGF and TGF-

beta

PDGF early stages HA synthesis and later sulfated

GAGs


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TGF-beta early Fn and later Type I

collagen, elastin and sulfated GAGs and;

inhibits collagen degradation by

Downregulation of collagenase gene

Upregulation ofTIMPs


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Angiogenesis in Granulation Tissue

Angiogenesis: The regeneration of new bloodvessels

From pre-existing ones

Crucial regenerative response in all injuredtissues whether the end result is scar tissue

formation or restoration of normal tissue

architecture

adequate supply of oxygen and nutrients

Common elements in fibrotic and

regeneration pathways to promote blood

vessel re eneration


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Low-O2 tension signal for angiogenesis

Initially hypervascularization reddish

app. during remodeling into scar tissue

extra apoptosis

Macrophages said to be involved in

apoptosis of endothelial and their

supporting cells - pericytes


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Reinnervation of granulation tissue

Peripheral sensory and sympatheticpostganglionic nerves - regenerate in healing

wounds

Play a significant role in the formation ofgranulation tissue because of their effects on

inflammation

W

ound 1-2 days degeneration of nervesdistal to injury

Next two weeks hyperinnervation of

granulation tissue


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Subsequent regression

Same pattern of advance and recession as

angiogenesis may be mechanisticallycoupled

Injured sensory and sympatheticpostganglionic nerves appear to help mediatethe inflammation phase (neurogenicinflammation) via antidromic (conduction inreversed direction) stimulation of

neuropeptides such as substance P mastcell activation and degranulation

Intimate microanatomic association of mastcells and nerves in dermis


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Injury to nerves degranulation of mast cells

releases histamine and cytokines

Vasodilation

Increased capillary permeability

Attraction of neutrophils and macrophages

Increased fibroblast activity


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Remodeling ofGranulation Tissue into Scar

Final phase of structural repair - granulationtissue remodeled into a relatively acellularfibrous scar tissue

The scar differs from normal dermis in severalways Fn and HA levels return to normal

Decorin PG is lower than normal skin,

Chondroitin-4-sulfate PG is much higher

Collagen organization uninjured dermis reticular app (basket-weave like)

repaired dermis type I collagen MMPs - cross-linkedby lysyl oxidase into thick bundles parallel to wound

surface


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MMPs produced by epidermis and fibroblasts

Elastin fibers reduced

Scarmaturation vascular and neural

network density normal

Reduction in fibroblast number by apoptosis ~ 6 months in humans

Tensile strength increases with degree of cross-

linking; but not as normal tissue

Feedback loops inhibitory factors to end

the repair process


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Wound contraction in dermal repair

Mammals with loose skin excisional woundclosure aided by contraction of the dermis

Decrease the area to be covered by dermis and

filled in by scar tissue Most importantly in rodents (~90%); lesser in

pigs and humans (50%)

Fetalmammals regeneration in absence ofdermal contraction

Development increase in contraction along

with decreased capacity for regeneration


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f


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Molecular comparison of wounded vs

unwounded skin

Transcriptional profiling

whole skin or fibroblasts

A study by Iyer 8600 gene profiling

Genes Transduction of serum signals Entrance and progression through cell cycle

Wound repair 10 with clotting and hemostasis

8 inflammation 6 re-epithelization

12 angiogenesis

19 remodeling of granulation tissue

200 novel genes with unidentified function


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Fetal skin heals without scarring

Fetal mammalian skin repair w/o scarring

after excisional wound

Less contraction

Rapid re-epithelization and fibroblast

migration

Granulation tissue lays normal ECM

architecture

Late gestation regeneration to adult

response of fibrosis


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Cellular and ECM differences

Collagen and noncollagen protein synthesis higher than normal in fetal and adult ratwounds

Ratio collagen/total protein in wounded adultskin > unwounded adult skin

In fetal skin no significant differencebetween wounded and unwounded skin

No excessive type I collagen in fetal skin andfibril organization in reticular fashion


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Fetal skin normal pattern of collagen

synthesis and architecture

Fetal wound fibroblasts synthesize higher

levels of HA and HA receptor better cell

movement

HA inhibits fetal fibroblast proliferation anddecrease scar formation

Sulfated PG synthesis does not accompany

collagen synthesis in fetal wounds Fetal skin higher type III/type I collagen

ratio

F l d i i l i fl


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Fetal wounds minimal inflammatory

response

Major difference fetal wounds minimal

inflammatory response

Development immune system response to

injury suppresses regeneration in favor of scar

tissue formation

Fewer platelets in fetal wounds

Macrophage no. and persistence lower than

in adult wounds


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Thus, types and proportions of cytokines and

GFs involved in inflammatory response

different in fetal and adult wounds

Lower levels ofPDGF, TGF-1 and 2 and their

receptors in unwounded and wounded fetal

rat skin TGF-beta3 higher in fetal than adult

In adults PDGF induces persistent exp of

IL-6 maintains an environment thatpromotes production and deposition of

fibrotic matrix


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Some studies intrinsic differences in fetaland adult fibroblasts result of differentiation

independent of maturation of immune system

Fetal fibroblasts unique phenotype differsin production of and response to GFs,

synthesis of matrix components, pericellularHA coats and antigenic determinents

Wound environment major determiningfactor

Extent of injury - Tatoos repair by noscarring total area large but smallerindividual injury no inflammatory response

repair of skin wounds by fibrosis

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