CONTINUING MEDICAL EDUCATION

Cutaneous scarring: Pathophysiology, molecularmechanisms, and scar reduction therapeutics

Part I. The molecular basis of scar formation

Christos Profyris, MA, BM, BCh (Oxon), MRCS (Eng),a Christos Tziotzios, MA, MB, BChir (Cantab),MRCP (UK),b and Isabel Do Vale, MB, BCh (Wits)c

London and Cambridge, United Kingdom; and Parktown, South Africa

Cutaneous scarring is often the epicenter of patient-related concerns, and the question ‘‘Will there be ascar?’’ is one that is all too familiar to the everyday clinician. In approaching this topic, we have reviewedthe pathology, the embryology, and the molecular biology of cutaneous scarring. ( J Am Acad Dermatol2012;66:1-10.)

Key words: cutaneous scar; early growth response protein-1; homeobox13; interleukins; mechanisms ofscarring; platelet-derived growth factor; transforming growth factorebeta; Wnt pathway.

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Target Audience:Dermatologists and others involved in the delivery of dermatologic care.

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Learning ObjectivesAfter completing this learning activity, participants should be able todescribe the 3 pathological stages of cutaneous scarring and delineatethe differences between embryonic regenerative healing and adult scar-induced healing.

Date of release: January 2012Expiration date: January 2015

" 2011 by the American Academy of Dermatology, Inc.doi:10.1016/j.jaad.2011.05.055

1

People with exaggerated skin scarring may facesubstantial physical and psychosocial conse-quences.1 In this first installment of a two-partcontinuing medical education series, we aim toexplore the pathophysiology underlying cutaneousscarring and the molecular mechanisms governingscar formation. Understanding the biology of scar-ring will allow for a betterunderstanding of the scien-tific basis of scar-reductionstrategies. The latter shall bediscussed in Part II of thisseries.

METHODOLOGYIn preparing this work, we

used PubMed to perform lit-erature searches on scar-related research. Key termsused in the search were‘‘scarring,’’ ‘‘wound healing,’’‘‘prevention,’’ and ‘‘treat-ment.’’ Review articles wereused as an initial source ofinformation and, where rele-vant, information from pri-mary research papers wasobtained.

PATHOPHYSIOLOGYOF THE CUTANEOUSSCARKey pointsd The inflammatoryphaseof wound healing aimsto contain the injury andprevent infection

d Theproliferativephase ischaracterized by granu-lation tissue—composedof macrophages, fibro-blasts, and epithelialtissue

d The remodeling phaseis the lengthy processof extracellar matrix re-organization around the site of injury

d Embryonic cutaneous wounds in the firstthird of gestation heal without a scar

Disruption of cutaneous epithelial continuity re-sults in a characteristic pathophysiologic response.This response has been traditionally subcategorizedinto the three phases of normal wound healing.These phases are the inflammatory, proliferative,

and remodeling phases (Fig1). Wound healing, however,is a dynamic process, and atany point in time, processesoccurring in one phase over-lap with those occurring inanother.2

Inflammatory phase(days 1-3)

After the disruption of ep-ithelial integrity, the immedi-ate priority is hemostasis.This is achieved by activationof the extrinsic clotting path-way. Ultimately, this resultsin formation of a fibrin he-mostatic plug, which is fur-ther solidified by the arrivalof platelets from the localmicrocirculation.2

Once the danger of exsan-guination subsides, the nextpriority is the removal of deadtissue and the prevention ofinfection. Inflammatory cellsare crucial to this process. Forthe first 5 days, neutrophilsenter the fibrin-rich zone ofinjury. Through their actionsof phagocytosis and proteasesecretion, neutrophils kill lo-cal bacteria and help degradedead tissue. On the third dayafter injury, macrophagesalso enter the injury zone. Inaddition to phagocytosingpathogens and tissue debris,these cells secrete amultitude

of growth factors, chemokines, and cytokines. These

CAPSULE SUMMARY

d After cutaneous injury, thepathophysiology of wound healing ischaracterized by an inflammatory phase(days 1-3), a proliferative phase (days4-21), and a remodeling phase (day 21 toyear 1).

d Mammalian cutaneous wounds duringthe first third of gestation do not scar,because healing occurs via tissueregenerative pathways.

d Scarring is a healing process that hasbeen selected for during evolutionbecause it tackles pathogens quickly,walls off foreign bodies, and seals off aninjured area from the environment.

d Transforming growth factorebeta(TGFb) is pivotal in scar-mediatedhealing.

d The expression of TGFb1 and TGFb2

enhances scarring, and the expression ofTGFb3 reduces scarring.

d The proinflammatory cytokinesinterleukin-6 (IL-6) and IL-8 augmentscarring, but the antiinflammatorycytokine IL-10 has the opposite effectson the scarring response.

d Homeobox b13, the Wnt signalingpathway, early growth response protein-1, and platelet-derived growth factor allpropagate a robust fibroblast responsein the healing wound, leading toincreased scarring.

From the Department of Plastic and Burns,a Chelsea and West-minster Hospital, London; Addenbrooke’s Hospital,b CambridgeUniversity Hospitals NHS Foundation Trust, Cambridge, UnitedKingdom; and the Department of Plastic and ReconstructiveSurgery,c Charlotte Maxeke Johannesburg Academic Hospital,Parktown, South Africa.

Reprint requests: Christos Tziotzios, MA, MB, BChir (Cantab), MRCP(UK), Addenbrooke’s Hospital, Cambridge University HospitalsNHS Foundation Trust, Hills Road, Cambridge CB2 0QQ,Cambridge, United Kingdom. E-mail: ct287@cantab.net.

0190-9622/$36.00

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signaling molecules are vital for the coordination ofdownstream events occurring during the proliferativephase.2

Proliferative phase (days 4-21)Granulation tissue—composed of macrophages,

fibroblasts, and endothelial cells—is the hallmark ofthe proliferative phase. This tissue replaces the fibrinhemostatic plug set up during the inflammatoryphase. Macrophages, via the secretion of platelet-derived growth factor (PDGF) and transforminggrowth factorebeta 1 (TGFb1), induce fibroblaststo proliferate and lay down type III collagen. Thisprovides a structural framework for endothelial cellsto proliferate and lay down new vessels by theprocess of angiogenesis.2

Reepithelialization is essential for the reestablish-ment of tissue integrity. Keratinocytes adjacent to thewound edge and in hair follicles undergo dediffer-entiation and reorganize their adhesion molecules.This loosens their connections to both the basementmembrane and to each other, and subsequentlyallows them to migrate across the wound surfaceand close the skin defect.2

Remodeling phase (day 21 to year 1)During the remodeling phase, formation of granu-

lation tissue ceases through apoptosis of the respon-sible cells. This process is important, because itsaberration leads tohypertrophic scarring andkeloids.2

Withmaturation of thewound, the composition ofthe extracellular matrix undergoes change. The typeIII collagen deposited during the proliferative phaseis slowly degraded and replaced with stronger type Icollagen. This type of collagen is oriented as smallparallel bundles, which differs drastically from thebasket-weave orientation of collagen present innormal dermis.2

Towards the later stages of healing, the woundundergoes a contractile response through the actionof myofibroblasts. By virtue of their multiple attach-ment points to collagen, these actin-rich cells con-tract and reduce the surface area of the scar.2

Macroscopic considerationsThe immediate macroscopic appearance of the

wound after injury is that of a skin defect with a

glistening surface attributed to the fibrin exudate. Afew days later, eschar—a collection of dead tis-sue—replaces this defect. In the coming weeks, ared and indurated cutaneous scar forms. The scarremodels in the year that follows to become softand slightly lighter in color than the surroundingskin.3,4

The abnormal architecture of collagen thatresults following the remodeling phase is thecause of the visible cutaneous scar. Within the

Fig 1. Histologic view of the wound healing process. (A)Inflammatory phase of wound healing (day 1). (B) Earlyproliferative phase of wound healing (day 5). (C) Lateproliferative phase of wound healing with early remod-eling (day 14). C, Clot; D, dermis; E, epidermis; ES,eschar; F, fatty tissue; G, granulation tissue; G/S, lategranulation tissue/early scar tissue; HE, hyperproliferativeepithelium; HF, hair follicle; M, muscle; RE, regeneratingepithelium. (Adapted from Schafer and Werner9 andreprinted with permission from the Annual Review ofCell and Developmental Biology, vol 23, " 2007 byAnnual Reviews [http://www.annualreviews.org].)

Abbreviations used:

EGR-1: early growth response protein-1IL: interleukinsPDGF: platelet-derived growth factorTGFb: transforming growth factor beta CI

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abnormal collagenous network, there is a notableabsence of hair follicles, sebaceous glands, andsweat glands. The extent to which this abnormaldermal phenotype arises depends on the depth ofinjury; deeper cutaneous injuries give rise to morescar tissue.5

Embryonic regenerative healingCutaneous wounds made during the first third of

gestation in mammals heal via tissue regeneration.This type of healing uses reactivation of the devel-opmental pathways that originally gave rise to thetissue. Ultimately, the original tissue constituents arereplaced and healing occurs without scar forma-tion.6 Consequently, this prompts the question,‘‘Why should there be a scar-mediated healingresponse?’’

In evolutionary terms, most wounds encoun-tered by animals were the result of postcombattrauma or falls. Consequently, the common woundsfaced by mammals were bites, blows, contusions,and degloving injuries. These injuries involvedwidespread areas of tissue and were frequentlycontaminated with bacteria and foreign bodies. Insuch a scenario, evolutionary pressure favored ahealing response that would control infection, walloff foreign bodies, and seal off the injured areafrom the environment. The inflammatory and pro-liferative phases witnessed in normal adult woundhealing are remnants of this evolutionary selection.The multitude of leukocytes in the inflammatoryphase helps prevent systemic infection, and thefibroblast activity during the proliferative phasewalls off foreign bodies and reestablishes tissueintegrity. In stark contrast to the scar-forming heal-ing response, regenerative healing—as witnessed inearly embryos and in liver regeneration—has anotable absence of fibroblast or inflammatory cellactivity. In an evolutionary context, this attribute ofregenerative healing would have been negativelyselected for, because animals would face a high riskof septicemia and death.7

Wound healing today is likely to take place at aninjury site that has been debrided, sterilized, andapproximated with the use of suture material. As aresult, the coarse and dirty wounds encounteredthroughout evolution are less of a commonality andthe abundance of inflammatory cells and fibroblastsseen in scar-mediated healing are less of a necessity.In fact, in such controlled conditions for woundhealing, a regenerative response would provide theadvantage of a superior cosmetic result and wouldavoid the potential for aberrant scarring as seen inhypertrophic scars and keloids.7

MOLECULAR BIOLOGY OF WOUNDHEALINGKey pointsd TGFb1 and TGFb2 expression leads to in-creased scarring, whereas expression ofTGFb3 reduces scarring

d Proinflammatory cytokines interleukin-6(IL-6) and IL-8 enhance scarring, whereasthe antiinflammatory cytokine IL-10 de-creases the amount of scar tissue

d Homeobox b13, the Wnt signaling pathway,early growth response-1, and platelet-derived growth factor all favor fibroplasia

The mammalian cutaneous wound is enrichedwith a multitude of extracellular matrix proteins,growth factors, and cytokines not normally presentin intact skin.8 Because the literature on moleculesproposed to have a role in wound healing is vast,the following account will concentrate on mole-cules that are noted to have a significant influenceon tissue scarring.9,10 Preference was given tomolecules with robust research evidence. Keymolecules in the wound healing machinery andtheir pathologic significance are summarized inTable I.

Transforming growth factorebetaSignaling. TGFb is crucial in the regulation of

wound scarring. The three isoforms, TGFb1e3,exert their effects by binding to dimeric TGFbreceptor complexes. Upon activation, this receptorcomplex phosphorylates SMAD2 and SMAD3 pro-teins, which subsequently form dimers withSMAD4. In its dimeric form, this SMAD aggrega-tion is able to translocate into the nucleus andfunction as a transcription factor (Fig 2). TGFsb1 and b2 activate the receptor complex andthereby downstream signaling, whereas TGFb3 isa receptor antagonist and thereby blocks signaltransduction.9,10

Expression. Expression studies have stronglyimplicated TGFbs in scarring, because TGFbs andtheir receptors are expressed prominently in adultscar forming wounds. Conversely, in nonscarringfetal wounds, their expression is only transient.11 Inaddition, fibroblasts from both hypertrophic scarsand keloids consistently overexpress proteins in-volved in TGFb signal transduction.12-16

Animal studiesGenetically engineered animal models have pro-

vided invaluable information about the involvementof TGFb signaling in cutaneous wound healing.Knock-out mouse animal models for TGFb1 have

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revealed a characteristic defect in healing during theproliferative phase: at 10 days after incisional injury,histologic analysis of the wounds reveals reducedgranulation tissue bulk and collagen deposition.17

Conversely, in a transgenic mouse model whereTGFb1 was overexpressed in keratinocytes, type Icollagen expression was significantly up-regulatedand scar tissue bulk increased.18

The downstream mediators of TGFb signalinghave also been the subject of intense investigation.Because SMAD3 is an important molecule in TGFbsignaling, SMAD3 knock-out mice have been createdto decipher its role in wound healing. These studiesshow that animals deficient for SMAD3 have en-hanced cutaneous healing with reduced depositionof scar tissue.19

Recent research indicates that the scar-inducingsignal transduction mechanism of TGFb1 is mediatedvia the Wnt pathway (Fig 3). The application ofexogenous TGFb1—a scar inducer—on the woundsof b-catenin conditional knock-out mice results inminimal scarring.20 In addition, the constitutive ex-pression of an active form of b-catenin in SMAD3knock-out mice results in scarring not normally seenin the animals with SMAD3 deficiency alone.20 Thesedata imply that TGFb1 induces SMAD3 to transcribeproteins that activate the Wnt pathway to inducescarring.

Table I. Key molecules in the wound healingmachinery and their pathologic significance

Molecule Function

TGFb1

and TGFb2

Key in the proliferative phase ofwound healing; promote signalingvia SMAD and Wntedependentpathways to enhance scarring

TGFb3 Receptor antagonist; reduces scarringIL-6 and IL-8 Proinflammatory cytokines expressed

immediately after cutaneous injury;recruit and activate inflammatorycells, thereby promoting scarring

IL-10 Antiinflammatory cytokine thatreduces scarring; it inhibits theinfiltration of neutrophils andmacrophages towards the woundsite and dampens the expression ofproinflammatory cytokines

Homeobox13 Transcription factor; absent from thescar-free healing wounds offoetuses; favors fibroplasia

Wnt signalingpathway

Aberrant transduction has beenimplicated as causative factor ofaggressive fibromatosis;hypertrophic scars and keloidsdisplay excessive signaling via theWnt pathway

PDGF Secreted by macrophages during theproliferative phase of woundhealing and induces fibroblasts toproduce type III collagen andexocytose osteopontin;overexpressed in hypertrophicscars and keloids

Osteopontin Extracellular glycoprotein thatenhances fibroplasia; connectsintegrins on cell surfaces tocollagen within the extracellularmatrix and promotes cell adhesionand cellular migration; abolition ofosteopontin reduces the traffickingof both inflammatory cells andfibroblasts and also leads to a largernumber of these cells dying byapoptosis

EGRe1 A zinc-finger transcription factor; up-regulates the expression of TGFb1

and PDGF, mediators of enhancedfibroplasia

EGR-1, Early growth response protein-1; IL, interleukin; PDGF,platelet-derived growth factor; TGFb, transforming growthfactorebeta.

Fig 2. The transforming growth factorebeta signalingpathway (see text for details).

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Transforming growth factorebeta modula-tion studies. Studies using strategies to alter theconcentration of TGFbs during healing have re-vealed that the adult scar-forming mechanism canbe altered. The application of neutralizing antibodiesto TGFs b1 and b2 in incisional rat wounds results in asignificant reduction of extracellular matrix deposi-tion and, by extension, subsequent scarring.21

Importantly, the enhanced availability of TGFb3 inthe healing environment—via the application ofrecombinant TGFb3—results in scar tissue reductionand a subepidermal collagen architecture that re-sembles that of normal skin (Fig 4).22

SummaryThe studies above underpin the pivotal role of

TGFb signaling in scar-mediatedhealing. TGFsb1 and

b2 promote signaling via SMAD and Wnt-dependentpathways to enhance scarring. Conversely, TGFb3

prevents signaling via these pathways and leads toscar reduction. Deciphering the role of TGFbsignaling in wound healing has provided fruitfulavenues for therapeutic intervention.

Inflammatory response modulatorsProinflammatory cytokines. Regenerative

healing has a notable absence of inflammatorycell activity. Consequently, mediators of the inflam-matory response have been a focus of investigationin studies aiming to curtail scarring.7 Bothinterleukin-6 (IL-6) and IL-8 are expressed imme-diately after cutaneous injury and continue to beexpressed at elevated levels for several days. Thesecytokines act to recruit and activate inflammatorycells. In stark contrast to adult wounds, there is onlytransient expression of these interleukins in fetalscar-free healing wounds.23,24 Collectively, theseexpression studies suggest that scar tissue deposi-tion may be reduced by dampening the proinflam-matory cytokine profile of healing wounds.Importantly, the administration of IL-6 to fetalhealing wounds results in scarring that would notnormally occur.24

Antiinflammatory cytokines. IL-10 is an anti-inflammatory cytokine. It inhibits the infiltration ofneutrophils andmacrophages toward the wound siteand dampens the expression of proinflammatorycytokines. The importance of IL-10 as a scar modu-lator can be seen in IL-10 knock-out mouse embryos.Cutaneous injury to these knock-out animals resultsin adult-like scar-mediated healing, which is notobserved in wild-type embryonic counterparts.25

The antiscarring role of IL-10 has been exploited ina recent clinical trial.

Inflammatory response transcriptionfactors. The modulation of the inflammatory re-sponse transcription factors in cutaneous injurymodels has also produced promising data. Knock-out mouse models for the PU.1 transcription factorlack macrophages and functional neutrophils.Incisional wounds in neonates with this geneticbackground revealed normal healing with absenceof any obvious scarring.26 This adds weight to thehypothesis that the inflammatory response in woundhealing is a remnant of evolution and in manyinstances counterproductive to the healing of inci-sional wounds.

FibroplasiaThe molecular biology behind fibroblast pro-

liferation and collagen deposition is central to thestudy of wound healing. Consequently, several

Fig 3. The Wnt signaling pathway. Binding of Wnt to theseven-transmembrane domain receptor fizzled leads to theactivation of dishevelled (Dsh). Once activated, Dshblocks the ubiquinating complex of axin, glycogen syn-thase kinase 3 (GSK3), and adenomatous polyposis coli(APC). This leads to reduced ubiquination and therebyproteosomal degradation of b-catenin. b-catenin is nowable to transclocate into the nucleus and induce transcrip-tion through its association with the T-cell factor (TCF) andlymphoid enhancer factor (LEF) transcription factors.

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transcription factors and growth factors havebeen implicated for the control of these twoprocesses.

Homeobox b13. The homeobox (hox) b13 tran-scription factor is notably absent from the scar-freehealing wounds of fetuses. Conversely, in adults,hoxb13 expression is consistently high.27

Consequently, in an attempt to recreate this fetalparameter in adult healing wounds, hoxb13 knock-out animals were engineered. In these animals,healing is strongly enhanced with increased woundtensile strength and reduced scarring (Fig 5).Structural analysis of the healed wounds in theseanimals revealed collagen architecture that resem-bles that of uninjured dermis. This implies thatswitching off hoxb13 is an important trigger toactivating regenerative healing.28

Wnt signaling pathwayAberrant transduction via the Wnt signaling path-

way has been implicated as a causative factor ofaggressive fibromatosis; a connective tissue disorderleading to multiple subcutaneous nodules—des-moid tumors—and an excessive scar-forming heal-ing response.29 Hypertrophic scars and keloids, inindividuals without this disorder, also display exces-sive signaling via the Wnt pathway (Fig 3).30 Theincidence of hypertrophic scars and keloids is higher

in certain body areas, and regional variation in theactivation of this pathway would be a reasonablespeculation—one that has not been addressed in thescientific literature.

In aggressive fibromatosis, there is a mutation ofthe b-catenin gene, which leads to stabilization ofthe catenin protein product and thereby continuousactivation of the Wnt signaling pathway. Transgenicmice that constitutively express this mutant form ofb-catenin develop a phenotype similar to aggres-sive fibromatosis. In addition, experimental cuta-neous injury in these animals gives rise to excessivescarring reminiscent of keloids and hypertrophicscars.31

In order to further assess the outcome of manip-ulating b-catenin levels during wound healing, aconditional knock-out mouse was engineered. Inthis animal model, the b-catenin gene could bespecifically deleted at the site of experimental injury.In comparison, wild-type mice, animals with theconditional deletion of b-catenin, display muchsmaller wounds and have fewer fibroblasts in theirgranulation tissue.20

Early growth response protein-1. Earlygrowth response protein 1 (EGR-1) is a memberof the zinc-finger transcription factor group. Uponcutaneous injury, its concentration within fibro-blasts rises acutely.32 In mouse embryos that have

Fig 4. The application of transforming growth factorebeta 3 (TGFb3) to wound surfacesstimulates scar-free healing. (A) Macroscopic and microscopic views of a placebo-treatedwound. There is a visible scar with an abnormally organized dermis. (B) Macroscopic andmicroscopic views of a recombinant TGFb3-treated wound. In comparison to the placebo-treated wound, scarring is not visible and the dermal architecture in the wound matches thesurrounding uninjured dermis. (Adapted from Ferguson and O’Kane7 with permission from theauthors and the Royal Society.)

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been modified to overexpress EGR-1, excisionalwounds result in enhanced collagen depositionand wound contraction. Because EGR-1 up-regulates the expression of TGFb1 and PDGF, thesegrowth factors are the likely mediators of thisresponse.33

Platelet-derived growth factor. PDGF exists inseveral isoforms, and signals via the activation ofdimeric transmembrane tyrosine kinase receptors.Within cutaneous wounds, it is a strong inducer offibroblast-mediated collagen deposition, and it hasbeen observed that both PDGF and its receptor aremarkedly overexpressed in hypertrophic scars andkeloids.34,35 Importantly, recent studies have shownthat PDGF secreted by macrophages during theproliferative phase of wound healing induces fibro-blasts to produce and exocytose osteopontin.36

Osteopontin is an extracellular glycoprotein thatconnects integrins on cell surfaces to collagen within

the extracellular matrix. This acts to promote celladhesion and enhance cellular migration. In addi-tion, via integrin-mediated intracellular signaling,osteopontin provides an important antiapoptoticsignal via the nuclear factor kB group of transcriptionfactors.37 Importantly, the application of osteopontinantisense oligonucleotides in mouse incisionalwounds results in accelerated healing with reducedgranulation tissue and scarring.36 These results sug-gest that the abolition of osteopontin from thewound environment reduces the trafficking of bothinflammatory cells and fibroblasts and also leads to alarger number of these cells dying by apoptosis.

CONCLUSIONAn increased understanding of the molecular

mechanisms governing regenerative cutaneous heal-ing would be expected to dispel the age-old notionthat scarring is an inevitable consequence of injury or

Fig 5. Homeobox13 (Hoxb13) knock-out (KO) incisional wounds show enhanced healing.Full-thickness hematoxylineeosin-stained sections from wild-type (WT) (A) and Hoxb13 KO(B) skin harvested 7 days after wounding. The wounded areas, outlined in black, indicate lessdeposition of scar tissue in Hoxb13 KO animals. Full-thickness Masson trichromeestainedsections of the dermis, emphasizing collagen structure, in wounded and uninjured skin of bothHoxb13 KO mice and WT animals. Hoxb13 KO mice wounds (D9) displayed a basket weavecollagen architecture similar to uninjured skin (D). In WT animals, collagen architecture tookthe form of small parallel bundles (C9) that differed from uninjured skin (C). (Adapted fromMack et al28 with permission.)

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surgery. Numerous approaches have been taken tosuccessfully manipulate the adult scar-formingwound environment in the context of dermatologicsurgery with the aim to recreate a scar-free healingenvironment. So, ‘‘Will there be a scar?’’ Part II willdiscuss the evidence-base of the available scarreduction modalities and attempt to answer thisquestion.

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