Epithelialization in Wound Healing: A Comprehensive Review

COMPREHENSIVE INVITED REVIEW

Epithelialization in Wound Healing:A Comprehensive Review

Irena Pastar,1 Olivera Stojadinovic,1 Natalie C. Yin,1 Horacio Ramirez,1

Aron G. Nusbaum,1 Andrew Sawaya,1 Shailee B. Patel,1

Laiqua Khalid,2 Rivkah R. Isseroff,3 and Marjana Tomic-Canic1,*1Wound Healing and Regenerative Medicine Research Program, Department of Dermatology and Cutaneous Surgery,

University of Miami Miller School of Medicine, Miami, Florida.2Department of Surgery, University of Miami Miller School of Medicine, Miami, Florida.3Department of Dermatology, UC Davis, Sacramento, California.

Significance: Keratinocytes, a major cellular component of the epidermis, areresponsible for restoring the epidermis after injury through a process termedepithelialization. This review will focus on the pivotal role of keratinocytes inepithelialization, including cellular processes and mechanisms of their regu-lation during re-epithelialization, and their cross talk with other cell typesparticipating in wound healing.Recent Advances: Discoveries in epidermal stem cells, keratinocyte immunefunction, and the role of the epidermis as an independent neuroendocrineorgan will be reviewed. Novel mechanisms of gene expression regulation im-portant for re-epithelialization, including microRNAs and histone modifica-tions, will also be discussed.Critical Issues: Epithelialization is an essential component of wound healingused as a defining parameter of a successful wound closure. A wound cannot beconsidered healed in the absence of re-epithelialization. The epithelializationprocess is impaired in all types of chronic wounds.Future Directions: A comprehensive understanding of the epithelializationprocess will ultimately lead to the development of novel therapeutic ap-proaches to promote wound closure.

SCOPE AND SIGNIFICANCEKeratinocytes, the major cellu-

lar component of the epidermis, arenot only important for barrier main-tenance but also for its restorationupon injury through a process knownas epithelialization. This reviewwill focus on cellular processes andmechanisms of their regulationduring re-epithelialization. Manycellular features important for re-epithelialization, including mecha-nisms of transcriptional regulation,such as microRNA (miRNA) andhistone modifications, as well asrecent discoveries regarding epider-mal stem cells (ESCs), immune, and

neuroendocrine functions of the epi-dermis will be discussed.

TRANSLATIONAL RELEVANCE

Epithelialization is defined as aprocess of covering denuded epithe-lial surface. The cellular and molec-ular processes involved in initiation,maintenance, and completion of epi-thelialization are essential for suc-cessful wound closure. A great deal ofresearch effort has been focused onunderstanding these processes inboth acute and chronic wounds. Re-cent advances in cell therapies forchronic wounds will also be reviewed.

Marjana Tomic-Canic, PhD

Submitted for publication April 22, 2013.

Accepted in revised form September 20, 2013.

*Correspondence: Wound Healing and Re-

generative Medicine Research Program, Depart-

ment of Dermatology and Cutaneous Surgery,

University of Miami Miller School of Medicine,

1600 N.W. 10th Avenue, RMSB, Room 2023-A,

Miami, FL 33136 (e-mail: [email protected]).

j 445ADVANCES IN WOUND CARE, VOLUME 3, NUMBER 7Copyright ª 2014 by Mary Ann Liebert, Inc. DOI: 10.1089/wound.2013.0473

CLINICAL RELEVANCE

Epithelialization is an essential component ofwound healing used as a defining parameter of itssuccess. In the absence of re-epithelialization, awound cannot be considered healed. Barrier breachprovides a portal for wound infection. This processis impaired in all types of chronic wounds. Failureof keratinocytes to maintain the barrier may con-tribute to wound reoccurrence, which is anothersignificant clinical problem. A better understand-ing of the epithelialization process may provideinsights for new therapeutic approaches to accel-erate wound closure.

DISCUSSION OF FINDINGSAND RELEVANT LITERATUREEpidermis and its role in barrier maintenance

Epidermis as a skin barrier. The epidermis is astratified epithelium composed of several layers ofkeratinocytes, which provides a physical barrierbetween the environment and the organism,thereby protecting it from external agents andpathogens, and limiting the loss of fluids. This in-tegument is continually maintained by keratino-cytes that switch from a proliferative state in thebasal layer to a differentiated state as they migratethrough the granular layer, and finally become theflattened dead cell remnants of the cornified layer(Fig. 1). However, keratinocytes are not only im-portant in maintaining the epidermis but also inrestoring it after injury.

In the basal layer, keratinocytes are prolifera-tive and characterized by an infrastructure com-posed of keratin (K) intermediate filaments, K5and K14. The cells move toward the surface tra-versing layers known as the spinous layer, granu-lar layer, and stratum corneum. As they ascend,they undergo the process of differentiation, char-acterized by the switch from synthesis of K5 andK14 in the basal layer to K1 and K10 in the su-prabasal layers (Fig. 1).1 Basal cells are attached tothe basement membrane (BM) through hemi-desmosomes and focal adhesions, and suprabasalcells attach to their neighboring cells throughdesmosomes, which must be disconnected to allowkeratinocyte migration during the epithelializa-tion process.2 Formation of the barrier also re-quires the delivery of lipids and proteins containedin lamellar granules (in the granular layer) to thestratum corneum interstices, and the formation ofhigh molecular weight polymers through thecrosslinking of cornified envelope proteins (lor-icrin, involucrin, filaggrin, and other peptides).3

During terminal differentiation and formation ofthe cornified envelope, keratinocytes also becomedehydrated and flatten into a polyhedron, referredto as the terminal corneocyte.4 Corneocytes areinterconnected by corneodesmosomes, cell junctionstructures comprising the adhesive protein cor-neodesmosin. The lipid layer, secured to the pro-tein structure around the corneocytes, is oftenreferred to as the ‘‘mortar’’ in a ‘‘brick and mortar’’analogy of the stratum corneum. It forms the water

Figure 1. Structure of the epidermis. (A) Schematic illustration of epidermis. Keratinocytes are the major cell population of epidermis important formaintaining a barrier formation during homeostasis as well as restoring it after the injury. Mitotically active basal layer is adjacent to the basement membrane(BM). Keratinocytes in the basal layer are characterized by keratin (K) 5, K14, and K15. As they differentiate, keratinocytes form suprabasal layers known as thespinous, granular, and cornified layer. Differentiated keratinocytes express K1 and K10. (B) Immunolocalization of K5 (green) and K10 (red) in human epidermis.Nuclei are visualized with 4¢,6-diamidino-2-phenylindole–DAPI (blue). ª 2005 Wiley. Modified with permission from Morasso and Tomic-Canic.55 To see thisillustration in color, the reader is referred to the web version of this article at www.liebertpub.com/wound

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barrier and serves to maintain the fluid balance inthe epidermis.5

Regulation of keratinocyte differentiation. Duringthe process of differentiation, the undifferentiatedkeratinocytes change into differentiated nondivid-ing cells as they migrate upward to eventually giverise to the cornified envelope. Three major MAPkinase pathways are involved in the process of dif-ferentiation, activated by multiple stimuli, includ-ing calcium influx, epidermal growth factor (EGF),and tumor necrosis factor (TNF).6 This differentia-tion cascade also uses various protein kinase C(PKC) isoforms. Studies have shown that conven-tional PKCs, which are calcium dependent, areinhibitory, whereas the calcium-independent non-conventional PKC isoforms stimulate keratinocytedifferentiation markers.7 The final step in differen-tiation is the activation of the proteolytic and nu-cleolytic activity, which leads to the destruction ofcellular organelles and DNA. The increased intra-cellular calcium leads to transglutaminase activa-tion and formation of the cornified envelope throughenvelope precursor crosslinking.4 Several structuralproteins, including loricrin, involucrin, and filag-grin, become covalently crosslinked by calcium-dependent transglutaminase, with final attachmentof insoluble lipids forming the outermost layer of theepidermis. Integral in maintaining the epidermalcalcium gradient and epidermal barrier is Scarf(skin calmodulin-related factor). Scarf acts as both,a calcium sensor, and a regulator of target proteinfunction in barrier maintenance and formation.8

Keratinocytes continually renew, traveling up-ward from the basal to differentiated layers. Acontinuous, dynamic renewal throughout homeo-stasis as well as during disruption of the epidermalbarrier is maintained by ESCs.9

The role of ESCs in wound healingThe regenerative capacity of the skin relies on

the local populations of ESCs. ESCs reside withinspecific microenvironments, stem cell niches,which regulate their activity and fate.10,11 Thereare three distinct ESC niches identified to date:bulge of the hair follicle (HF), the base of the se-baceous gland, and the basal layer of the inter-follicular epidermis (IFE) (Fig. 2).12,13 Undernormal homeostatic conditions, each discrete ESCniche behaves unipotently, replenishing its ownrespective tissue compartment. It has been pro-posed that within the IFE, a hierarchy exists,comprising slow cycling stem cells in the basallayer, which produce a population of transitamplifying cells that undergo a limited number ofdivisions before differentiating as they ascendthrough the suprabasal layers.14 However, an al-ternative model has been put forth in which a sin-gle progenitor has the ability to divide into eithertwo undifferentiated basal cells, two terminal dif-ferentiating cells, or one of each type.14–16 Recentevidence supports the notion of a hierarchy andsuggests that it is the slow-cycling cells of theIFE and not the transit amplifying cells thatare involved as major contributors to long-termtissue repair.17

Figure 2. Regenerative capacity of the skin relies on local populations of epidermal stem cells. (A) Schematic representation of a hair follicle (HF) with themultipotent stem cells (red). Three distinct epidermal stem cell (ESC) niches are identified so far: bulge of the HF, the base of the sebaceous gland (SG), and thebasal layer of the interfollicular epidermis. It has been shown that stem cells migrate from the HF and interfollicular stem cell niche to aid repair andepithelialization upon skin wounding.18–21 ESCs generate transit amplifying cells, which will differentiate to form the stratified epidermal layers. (B) Section ofhuman skin stained with hematoxylin and eosin to distinguish epidermis (E) and dermis (D).ORS, outer root sheet; IRS, inner root sheet. To see this illustration incolor, the reader is referred to the web version of this article at www.liebertpub.com/wound

KERATINOCYTES IN WOUND CLOSURE 447

In response to epidermal injury, both the HF andIFE niches participate in re-epithelialization of thewound defect.18–21 Ito et al. demonstrated that in amurine full-thickness wound model, HF bulge stemcells characterized by the expression of K15 mi-grate into the IFE initially postwounding. How-ever, the contribution of K15-positive cells may betemporary, as evidenced by their absence from theIFE several weeks after healing.18 Interestingly,studies utilizing more recent bulge stem cellmarkers, such as LGR522 and SOX9,23 indicate thepresence of bulge-derived ESCs in the repairedepidermis long after healing has taken place. Therole of HF ESCs was further defined by Langtonet al. who demonstrated a delay in the earlyphase of re-epithelialization when acute incisionalwounds were created in HF-deficient mice.20

Complete closure, however, was achieved presum-ably through recruitment of ESCs from the IFEniche. These findings suggest that in incisionalwounds characterized by a minimal epidermal de-fect, HF ESCs provide an initial burst in the rate ofhealing, but are likely less important than in largerwounds, where re-epithelialization must occurover extensive surface areas.

The role of ESCs in wound healing as well as themechanism, which orchestrate their function, hasbeen studied predominantly in mouse modelspointing to the need for confirmatory evidence inhuman skin. Further insight into the wound heal-ing process will help focus research efforts to elu-cidate cellular defects contributing to nonhealingwounds aiming to improve current and developingnovel stem cell-based therapies for wound healingdisorders.

In addition to ESCs, somatic cell therapy isutilized to stimulate wound closure. These tissue-engineered human skin equivalents are FDA ap-proved for the treatment of chronic wounds. It hasbeen suggested that this cell therapy promoteshealing through the release of various cytokinesand growth factors when applied to a nonhealingwound after surgical debridement.24 However, arecent study documented that even without tissueengineering, human allogeneic keratinocytes andfibroblasts in a spray formulation can be success-fully used as a therapy for nonhealing venousulcers.25 Further insights into the mechanism ofaction of cell-based therapeutics are needed forbetter understanding and to determine the opti-mal use. A clinical trial to assess the added ad-vantage of viable cells within the bioengineeredconstructs as compared with the matrix alonein the treatment of chronic wounds is currentlyongoing.26

Keratinocyte migration and proliferationduring epithelialization

Upon acute skin injury, as the barrier is dis-rupted, neutrophils, monocytes, and macrophagesare recruited to the site of injury.27 Subsequently,keratinocytes become activated, and the activationprocess is achieved by expression of several cyto-kines and growth factors. The activated phenotypeis marked by changes in the cytoskeleton networkand cell surface receptors essential for re-epitheli-alization, namely, expression of K6 and K16, al-lowing keratinocytes to migrate into the wound tofill the defect.28

Keratinocyte migrationTo close the defect in the epidermis, keratino-

cytes at the wound edge must first loosen theiradhesion to each other and to the basal lamina, andneed to develop the flexibility to support migrationover the freshly deposited matrix. This processis modulated sequentially beginning with disas-sembly of cell–cell and cell–substratum contactsmaintained through desmosomes and hemidesmo-somes, respectively.29 This release allows kerati-nocytes to start migrating from the wound edgeover the denuded area, whereas keratinocytesbehind the migrating tongue begin to proliferate(Fig. 3).30 Numerous regulators play a critical rolein modulating the proliferation and migration ofkeratinocytes during epithelialization.

For the process of migration to begin, the cell–cell interaction needs to be dissolved. During thisprocess, PKCa gets activated and leads to the con-version of calcium-independent to calcium-dependentdesmosomes, thus decreasing their adhesive prop-erties.31 Among multiple transcription factors in-volved in this process, the transcription factor Slugallows for additional release of keratinocytes byincreasing desmosomal disruption.32 Hemidesmo-somes linking the BM to the basal layer of kerati-nocytes need to be disassembled as well to allow formigration.33 The a6b4 integrin expressed by basalkeratinocytes binds to an isoform of laminin calledlaminin-5 (LN5) in the lamina densa of the BM,contributing to the adhesive property of hemi-desmosomes. One proposed mechanism suggeststhat there is a differential affinity of integrinsto laminin LN5, namely, the precursor form,nonproteolytically processed LN5. The nonproteo-lytically processed form has a major bindingaffinity for a3b1, while the processed form bindspreferentially to a6b4. When keratinocytes startmigrating, a switch from a6b4 to a3b1 integrin forLN5 binding occurs.34 The inside-out modulationof a6b4 affinity for ligands by alterations of the

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cytoplasmic portion of integrins is another proposedmechanism involved in keratinocyte migration;35

namely, serine phosphorylation of b4 subunitsthrough action of PKCa increases keratinocyte mo-tility by increasing disassembly of hemidesmosomes,while EGF and macrophage-stimulating proteinfound in the wound environment modulate thisPKCa-dependent phosphorylation.36 Migrating ker-atinocytes show an upregulation of K6, K16, andK1737 keratins, which are hypothesized to increaseviscoelastic properties of migrating cells,38 and theirexpression is regulated by growth factors presentwithin the wound environment.

Multiple regulators such as growth factors andcytokines, integrins, keratins, matrix metallopro-teinases (MMPs), chemokines, and extracellularmacromolecules modulate keratinocyte migra-tion.27,39 Epidermal growth factors such as HB-EGF, EGF, and TGF-a, transactivate EGFR, which

directly stimulates keratinocyte migration andproliferation27 and induces expression of K6 andK16.40 On the other hand, our laboratories haveshown that glucocorticoids (GCs) can block EGF-mediated migration by repressing transcription ofK6/K16.40 EGF signaling is not properly executedin the epidermis of chronic wounds. In contrast tomembranous localization in normal epidermis, EGFRis primarily localized in the cytoplasm of keratino-cytes of the chronic wound.41 The carbohydrate-binding protein, galectin-3, may play a critical rolein the cytoplasmic localization of the EGFR, as ourlaboratories have demonstrated that in mice ge-netically depleted of galectin-3, wound healing isdelayed and the EGFR is localized intracellularlyin wound-edge keratinocytes, as opposed to nor-mal, membranous localization in wild-type animalswith normally healing wounds.42 In addition, non-specific disintegrin and metalloprotease 12 (ADAM12)

Figure 3. Epidermal keratinocytes migrate over the wound bed to epithelialize the wound gap. Immunofluorescence staining with keratin 17 (K17, red)antibody demonstrates epithelialization process in human ex vivo wound model. White arrows indicate wound edges after initial wounding, while yellow arrowspoint at the edges of the migrating epithelial fronts. K17 is not present at the time of wounding (0 h, A). Immediately after injury (A), keratinocytes releaseproinflammatory cytokines and growth factors, including interleukin 1 (IL-1), tumor necrosis factor a (TNFa), and epidermal growth factor (EGF). In response tothese stimuli, keratinocytes become activated and start migrating over the wound bed. Migrating keratinocytes show an upregulation of K17 (48 h, B). StrongK17 staining persisted over 4 days after the wounding when the wound is completely closed (96 h, C). A well-balanced communication with other cell types,fibroblasts, neutrophils, endothelial cells, monocytes, and macrophages (schematically presented at the bottom), B) through various cytokines and growthfactors (KGF, PDGF-bb, VEGF, GM-CSF, TGFb, IL-8), is necessary for successful epithelialization. Nuclei are visualized with DAPI (blue). White dashed linesindicate the dermal–epidermal boundary. KGF, keratinocyte growth factor; PDGF-bb, platelet-derived growth factor bb; VEGF, vascular endothelial growthfactor; TGFb, transforming growth factor b; GM-CSF, granulocyte–macrophage colony-stimulating factor; IL-8, interleukin 8. To see this illustration in color, thereader is referred to the web version of this article at www.liebertpub.com/wound


implicated in inactivation of HB-EGF- and IGF-binding proteins has been found induced in chronicwound epidermis.43 Together, this suggests thatone major impediment to healing of a chronicwound is the inability of the wound edge kerati-noctyes to respond to EGF family members, even ifsupplied externally. This may explain the clinicalfailure of topically applied EGF to improve healingin some human chronic wounds.44

Another group of growth factors, fibroblastgrowth factors (FGF) -2, FGF-7, FGF-10, and FGF-22 have also been shown to stimulate epithelial-ization mostly through paracrine effects.45 As anexample, FGF2 (also known as KGF) is producedby fibroblasts and acts in a paracrine fashionthrough the KGFR2IIIb receptor found exclusivelyon keratinocytes, resulting in increased migrationand proliferation during wound healing.45 Cyto-kines, such as IL-1, IL-6, and TNF-a, can alsomodulate migratory phenotypes of keratinocytes.Among its multiple functions during wound heal-ing, IL-1 increases secretion of FGF-7,46 whereasIL-6 acts through the STAT3-dependent pathway,which allows keratinocytes to respond to mitogenicfactors and stimulate migration.47 TGFb1 can alsopromote keratinocyte migration by stimulatingMMPs.48 For the wound to heal successfully, ker-atinocytes should be able to not only detach fromthe underlying basal lamina but also to move andmigrate through the fibrin and newly synthesizedextracellular matrix (ECM) of the wound, a processfacilitated by MMPs. Involved MMPs are MMP-1,MMP-2, MMP-3, MMP-10, MMP-14, MMP-19, andMMP-28.49,50 MMP-1, which is expressed abun-dantly at the wound edges, sustains keratinocytemigration on type 1 collagen, mediated by the a2b1integrin.51 Cytokines and growth factors secretedduring the wound healing process also stimulateMMP production. More importantly, optimalkeratinocyte migration during wound closure isdependent on tight regulation of MMPs and tissueinhibitors of metalloproteinases (TIMPs),52 whereasderegulated MMP/TIMP ratios demarcate chronicnonhealing wounds.53 Additional stimuli indepen-dent of the ECM, growth factors, and cytokines,such as electric fields, may also participate in woundhealing by directing cell migration.54 In intact hu-man skin, current is limited by the very high resis-tance of stratum corneum. Disruption of epidermalintegrity triggers the immediate formation of anendogenous electric field. A net flow of currentthrough the low-resistance wound pathway occursupon injury and results in the generation of a lateralelectric field within or beneath the adjacent epider-mis, thus contributing to keratinocyte migration.54

Keratinocyte proliferationUpon the advancement of migrating epithelial

tongue, the first layer that covers the wound, ker-atinocytes start to proliferate to ensure an ade-quate supply of cells to encase the wound. Theregulation of keratinocyte proliferation is depen-dent on the availability of growth factors, degree ofcell differentiation, and cell attachment to thesubstrate. Only the basal keratinocytes have theability to proliferate, while the terminally differ-entiated keratinocytes in the suprabasal layer losethis ability.55

Growth factors that play a major role in theproliferative process during epithelialization in-clude previously mentioned HB-EGF, EGF, TGFa,and KGF.56 Another growth factor present inwounds, insulin-like growth factor (IGF)-1, wasshown to act synergistically with HB-EGF instimulating keratinocyte proliferation.57 Increasedkeratinocyte proliferation has also been docu-mented in transgenic mice overexpressing epider-mal granulocyte–macrophage colony-stimulatingfactor, and this growth factor has been shown toaccelerate wound closure.58 MMPs, components ofthe ECM, and integrins, can work together to assistgrowth factors in promoting keratinocyte prolifer-ation. MMPs work through their proteolytic activ-ity to release growth factors from the wound matrixand they can also digest latent forms of growthfactors, such as IGF-1, converting them to activeforms.59 The ECM can also engage integrins tomodulate growth factor receptor pathways, leadingto an increase in growth factor activity.60 In sum-mary, it is the synergy between growth factors, theECM, and integrins that plays a pivotal role inregulation of keratinocyte proliferation during re-epithelialization. After being activated to repair aninjury, ultimately, the deactivated keratinocytesreturn to their normal differentiation pathway.Once the wound is healed, defined as being fullyepithelialized with no drainage, and covered by akeratinocyte layer, the proliferation signals ceaseand the stratification process begins.

Proliferation and migration of keratinocytes in thechronic wound. Keratinocytes at the nonhealingedges of chronic wounds are different both pheno-typically and biologically than keratinocytes com-prising intact epidermis or the edge of acutewounds. In contrast to normal skin, where mitoti-cally active keratinocytes reside only in the basallayer, keratinocytes in chronic wounds undergodivisions throughout the suprabasal layers. Thishyperproliferative epidermis is a result of c-mycactivation and overexpression.61 Parakeratosis

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(presence of the nuclei in the cornified layer) andhyperkeratosis (a thick cornified layer) are addi-tional properties characterizing chronic woundkeratinocytes61 (Fig. 4). Specific epidermal mor-phology can also be explained as a consequenceof deregulation of differentiation and activationpathways.62 K1/K10 and a subset of small proline-rich proteins, together with the late differentiation

marker filaggrin, were found to be suppressed,whereas late differentiation markers involucrinand transglutaminase 1 were induced in venousulcers when compared with healthy skin. The epi-dermis of chronic nonhealing wounds has reducedexpression of the precursor of the a3 chain ofLN5,63 contributing to impaired migratory capac-ity of these cells. Furthermore, b-catenin is presentin the nuclei of chronic wound keratinocytes evenin the cornified layer.61,64 All these factors yield tononmigratory, hyperproliferative epidermis thatfails to re-epithelialize and restore the barrier.

Cross talk between keratinocytes and othercell types during epithelialization

Complex interactions and cross talk betweenkeratinocytes, fibroblasts, endothelial, immunecells, and other cell types during all three phases ofwound healing are critical for successful woundclosure and repair (Fig. 3). Upon injury, keratino-cytes located immediately adjacent to the woundsite undergo an activation process in which theyalter their gene expression rendering them com-petent for migration over the wound bed. Onceactivated, keratinocytes produce signaling mole-cules that act in an autocrine and paracrine fash-ion, resulting in pleiotropic effects on multiple celltypes. These cells, in turn, respond by producingseveral signaling molecules that regulate kerati-nocyte activation during wound closure.

The initial phase of healing results in a release ofprestored IL-1 by keratinocytes. IL-1 functions inan autocrine fashion by increasing keratinocytemigration and proliferation, and by inducing theexpression of K6 and K16 in keratinocytes mi-grating into the wound.65 In addition to acting onthe keratinocytes, IL-1 activates nearby fibroblastsand increases the secretion of KGF, which in turnpromotes keratinocyte migration and proliferationas discussed above,45 and triggers the inflamma-tory cascade. The other common initiator ofkeratinocyte activation is the proinflammatorycytokine, TNFa.65 Similar to IL-1, TNFa can alsoact in an autocrine fashion to stimulate keratino-cyte migration, and in a paracrine fashion acti-vating fibroblasts and increasing the secretionof proteins of the FGF family of signaling mole-cules.66 This promotes fibroblast migration and de-position of ECM components conferring increasedkeratinocyte motility during re-epithelialization.67

Another important signaling molecule produced byboth keratinocytes and fibroblasts is TGF-b. Its ex-pression is upregulated upon wounding and has beenshown to induce granulation tissue formation andmyofibroblast differentiation facilitating contraction of

Figure 4. Epidermis of chronic wounds shows specific morphology thatdiffers from epidermis of healthy skin. Healthy human skin is composed ofseveral layers of keratinocytes. These cells lose their nuclei through the pro-cess of differentiation and form enucleated cornifed layer. However, epidermisof chronic wounds, such as pressure ulcers, venous ulcers, and diabetic footulcers, has distinct morphology. They are characterized by hyperproliferativeepidermis as a result of c-myc activation and overexpression.61 In addition,presence of hyperkeratosis (a thick cornified layer) and parakeratosis [pres-ence of the nuclei in the cornified layer (black arrow)] are additional hallmarksportraying epidermal keratinocytes of chronic wounds.61 To see this illustrationin color, the reader is referred to the web version of this article at www.liebertpub.com/wound


the collagen matrix and wound closure.68 In addition,TGF-b acts as an important regulator in reverting theactivated keratinocytes to the basal cell phenotype byinducing the basal cell-specific markers K5 and K14and reducing proliferation.69 However TGF-b signal-ing is suppressed in the epidermis of venous ulcers,70

which together with additional mechanisms41 con-tributes to the nonhealing and nonmigratory pheno-type of chronic wound keratinocytes.

The early response to wounding also results inthe release of chemokines by keratinocytes that actas chemoattractants for migration of immune cellsto the site of injury. Neutrophils arrive at thewound site within minutes of wounding and be-come the predominant cells in the wound for thefirst 2 days after the injury occurs. Neutrophils andplatelets entrapped and aggregated in the bloodclot release a wide variety of factors that amplifythe aggregation response, initiate a coagulationcascade, and/or act as chemoattractants for cellsinvolved in the inflammatory phase.71 Amongother proinflammatory cytokines, IL-6 is producedby neutrophils and has been shown to be importantin initiating the healing response; namely, IL-6 hasboth mitogenic and proliferative effects on kerati-nocytes and, at the same time, acts as a chemoat-tractant for neutrophils.47 The inflammatory phaseof wound healing continues with active recruit-ment of macrophages from blood vessels, which isorchestrated by growth factor signals from kerati-nocytes, and by foreign epitopes of microorgan-isms.72 The macrophage chemoattractant protein(MCP-1), a member of the CC family of chemo-kines, is induced in keratinocytes and promotesmigration of both macrophages and T cells to thesite of injury.73 The significance of this chemokineis underscored in MCP-1-deficient mice in whichre-epithelialization was significantly inhibited.74

Signals released by keratinocytes and fibroblastsupon the wounding target also skin endothelial cells(ECs). Growth factors, cytokines, and cell–cell andcell–matrix interactions activate ECs. ActivatedECs, platelets, macrophages, and fibroblasts releaseproangiogenic cytokines and growth factors, leadingto the invasion and migration of ECs into the ECM,EC proliferation, and new immature vascular for-mation.75 Early events in angiogenesis, particularlyEC migration and proliferation, are promotedby vascular endothelial growth factor (VEGF) andthe initial phase of wounding results in release ofVEGF by platelets in response to hypoxia.76 VEGFacts in a paracrine fashion not only on ECs but alsoon keratinocytes and immune cells promoting re-epithelialization and at the same time stimulatingangiogenesis and restoring oxygen perfusion.77

Taken together, growth factors and cytokinescontrol wound healing events, and the balanceof tightly regulated ligands and their receptorscoordinates progression through all phases ofhealing.27,39 However, nonhealing wounds fail toprogress and often remain in a prolonged inflam-matory phase. As a consequence, imbalances inwound proteases and their inhibitors in chronicwounds may cause degradation of growth factorsand cytokines in the wound site, inhibiting pro-gression of the healing process.53 Recent advancesin the field of miRNAs and histone modificationshave revealed new aspects and an additional levelof complexity regarding the molecular mechanismscontrolling normal and impaired wound healingprocesses, as detailed below.

Mechanisms of gene expression regulationduring epithelialization

Role of miRNAs in wound healing. miRNAs aresmall noncoding RNAs that regulate gene expres-sion at the post-transcriptional level by binding tothe 3¢ UTR of mRNAs, repressing their translationor causing messenger degradation.78 miRNAs aregenerated by sequential processing of long RNApolymerase II transcripts by two key RNase IIIproteins, Drosha and Dicer.79 To target mRNA,miRNA binds mRNAs in a unique manner usingWatson–Crick base pairing through a conserved 6-to 8-bp seed sequence. Importantly, one miRNAcan target hundreds of genes, whereas a singlegene can be targeted by multiple miRNAs.78,79

The importance of miRNAs in epidermal devel-opment and adult skin stem cell maintenance hasbeen established by experiments involving condi-tional depletion of either Dicer1 or Drosha in mu-rine epidermis.80,81 When either one of these twogenes were ablated in the epithelium, the barrierfunction of the skin was compromised.80,81 Fur-thermore, these studies identified several miRNAsthat were expressed differentially or exclusively inmurine epidermis as compared with other skinlineages. The miR-200 family (a, b, and c), miR-141,miR-429, and the miR-19/miR-20 family (miR-19b,miR-20, miR-17-5p, and miR-93) were preferen-tially expressed in epidermal lineage, while themiR-199 family was exclusively expressed inHFs.80,82 More recent studies compared the miR-NA expression patterns of in vitro differentiatedkeratinocytes, with miRNA profiles of ESCs, tran-sit amplifying keratinocytes and terminally dif-ferentiated keratinocytes isolated from humanskin.83 The results revealed eight upregulatedmiRNAs in differentiated keratinocytes (miR-23b,miR-95, miR-210, miR-224, miR-26a, miR-200a,

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miR-27b, and miR-328), and one downregulatedmiRNA (miR-376a) both in vivo and in vitro,suggesting their involvement in the process ofepidermal differentiation.83

Based on the multicellular nature of such acomplex process that depends on timed responseand signaling control, it was reasonable to assumethat miRNAs have a role in controlling woundclosure.84 This hypothesis was confirmed by sev-eral recent discoveries.85–88

miR-203, a very abundant miRNA that targetsp63 and contributes to barrier formation, was re-cently associated with wound healing.89,90 miR-203is downregulated at the migrating epithelium ofacute wound edges allowing the expression of p63,RAN (member of the G-protein superfamily), andRAPH1 (lamellipoidin), hence contributing to re-epithelialization.91 In contrast, the epidermis ofnonhealing chronic wounds has elevated miR-203levels.85 Another recent report has shown anantiproliferative effect of miR-483-3p in humankeratinocytes, induced by wounding in vitro andin vivo.92

The mechanisms by which deregulation of miR-NAs contribute to the pathogenesis of chronicwounds have also been proposed. A set of miRNAs,miR-16, miR-20a, miR-21, miR-106a, miR-130a,and miR-203 were found to be upregulated in thehyperproliferative nonmigratory epidermis ofchronic venous ulcers (Fig. 5).85 Furthermore, miR-130a and miR-21 inhibited epithelialization in vivoand ex vivo by targeting multiple genes importantfor wound closure, including the early growth re-sponse factor 3 (EGR3) and leptin receptor (LepR).85

Hypoxia-induced miR-210 has also been linked todecreased re-epithelialization in ischemic wounds.86,88

Together, these studies demonstrate that miRNAscontribute to both normal epithelialization and itsinhibition in nonhealing wounds.

Epigenetics of the wound healing process. Dif-ferent types of epigenetic regulation include dif-ferential methylation and hydroxymethylation ofDNA, histone modifications, ATP-dependent chro-matin remodeling, and higher order chromatinremodeling and positioning within the nuclear

Figure 5. Role of miRNAs in epithelialization. Schematic representation of miRNA biogenesis: miRNAs are transcribed in the nucleus as 70-bp precursorproducts that are processed into the mature *22-bp products by the Drosha and Dicer cytoplasmic enzymes. The mature miRNA interacts with the RNA-induced silencing complex (RISC) and binds to complementary sequences in target messenger RNAs (mRNA) leading to downregulation of gene expression.Suppression of miR-203 and induction of miR-483-3p promote keratinocyte migration during normal wound healing process. Induction of miRNA—203, -130a,-106a, 21, -20a, -16 in keratinocytes of nonhealing edges of chronic wounds aids to their pathogenesis by inhibiting epithelialization and wound closure. To seethis illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/wound


space. Each of these was found to participate inkeratinocyte differentiation and barrier formation(reviewed in Botchkarev et al.93) although theirinvolvement during wound healing and epitheli-alization remains mostly unexplored. Reduction ofhistone H3 lysine 27 trimethylation (H3K27me3)occurs at the healing edges of full-thicknesswounds in mice.94 H3K27me3 is a silencing chro-matin modification that can be set by the PolycombRepressive Complex 2 (PCR2)95 or removed by thespecific H3K27 demethylases Jmjd3 and Utx.96

Consistently, the components of the PCR2/Eed-Ezh2 complex were downregulated at the woundedges, whereas Jmjd3 and Utx were upregulated.94

In addition, less Eed was bound to the promoterregions of two genes important for wound healing,EGFR and myc, up to 3 days postwounding sug-gesting the existence of tight epigenetic regulationof the epithelialization process.94 Polycomb-group-mediated H3K27me3 also plays an important rolein balancing HF stem cell quiescence.97

Moreover, nuclear epigenetic changes andmiRNA regulation of gene expression seem to beinterconnected as miRNA expression can be regu-lated by histone modifications and DNA methyla-tion. Conversely, miRNAs can target epigeneticregulators such as DNA methyltransferases andhistone modifying proteins.98 For example, the re-epithelialization-related miR-203 is found to besilenced by DNA methylation of its promoter regionin several types of tumors resulting in induced cellgrowth and invasion.99 Furthermore, miR-203 canalso target Bmi-1, a component of the polycombrepressor complex 1 involved in migration and in-vasion of cancer cell lines.100 In addition, miR-205overexpression, which targets lipid phosphataseSHIP2 and increases keratinocyte migration dur-ing wound healing,101 has been found to suppressthe expression of Ezh2 (enhancer of zeste homolog2) in prostate cancer, suggesting a possible contri-bution to histone modifications.102

These new mechanisms of gene expression reg-ulation during wound closure provide novel in-sights and additional levels of complexity to thewound healing process. Further exploration ofthese mechanisms may help us to understand hownormal epithelialization occurs and why in chronicwounds this control is lost.

Epidermis as a neuroendocrine organAs a sophisticated physical barrier exposed to a

diverse range of stressors, the skin and epidermisin particular, have evolved as an independentneuroendocrine organ to efficiently sense and ap-propriately respond to the stress and changes in

the external environment. The dynamic capabilityof skin to communicate with the central nervoussystem through local production and systemic re-lease of hormones, neuropeptides, neurotransmit-ters, and biological regulators establishes it as aneuroendocrine organ.103,104 This interdependentcross talk involves expression of specific receptors,synthesis, activation or inactivation of the hor-mones, and exertion of biological activity, whichhelp maintain local and consequently systemichomeostasis.103 Both keratinocytes and melano-cytes share analogous properties with secretoryneurons by expressing a highly organized hypo-thalamus–pituitary–adrenal axis, which includescorticotropin-releasing hormone, urocortin, pro-opiomelanocortin, with its products adrenocortico-tropic hormone, a-melanocyte-stimulating hormone,and b-endorphin.105 In response to stress, skin cellscan also produce vitamin D3,106 cortisol,107 para-thyroid hormone-related protein,108 precursors tobiogenic amines,109 and the neurotransmitters—catecholamines and acetylcholine (Table 1).104,110

Furthermore, the skin is a site for activation ofsteroid hormones such as converting T4 to T3 ortestosterone to either 5a-dihydrotestosterone or es-tradiol.111 The production of these locally synthe-sized hormones and expression of their receptors iseither constitutive or can be induced by specificstimuli.107,112,113

The most prevalent cellular components of theneuroendocrine system in skin are keratinocytes,which not only express a vast repertoire of hormonereceptors, but can also synthesize hormones toenhance or impair wound healing. Most impor-tantly, keratinocytes possess the ability to syn-thesize cholesterol de novo, the precursor to allsteroids.114 Hormones as well as intermediates inthe cholesterol synthesis pathway, such as farnesylpyrophosphate (FPP), affect epithelialization.FPP acts as an agonist for the GC receptor, thusinhibiting keratinocyte migration and wound clo-sure.115 Sex steroid hormones are important forcontrolling skin turnover, and a large percentage ofandrogens and estrogens are synthesized locallyfrom inactive precursors.116 It has been shownthat estrogen has a mitogenic effect on keratino-cytes and increases the rate of epithelializationpostwounding in animal models.117 Exogenoussystemic or topical estrogen treatment enhanceshealing by stimulating matrix deposition and re-ducing inflammation.117 On the other hand, tes-tosterone possesses an inhibitory effect on skinrepair and epithelialization.118 These results donot imply differences in the healing rates betweenmales and females, but rather indicate the role of

454 PASTAR ET AL.

sex hormones in fine-tuning the wound healingprocess. Keratinocytes also express enzymes suchas steroid 11 b-hydroxylase (CYP11B1) essentialfor cortisol synthesis and 11b-hydroxysteroiddehydrogenase 1 (11bHSD1), which catalyzes con-version of inactive to active cortisol.107,119 Inhibi-tion of skin-specific GC synthesis by CYP11B1inhibitor metyrapone, accelerates wound closurein vivo.107 In addition, inhibition of 11bHSD1 leadsto increased proliferation of keratinocytes.119 In-hibition of GC synthesis in keratinocytes resultedin increased expression of proinflammatory IL1-bimplying that skin-specific cortisol synthesisserves as a local negative feedback to prevent ex-cessive inflammation upon initial injury and servesto regulate epithelialization.107

Another type of hormone, the catecholamineepinephrine, can impair keratinocyte migration ina dose-dependent manner by binding to the beta2-

adrenergic receptor (beta2AR) leading to delayedepithelialization.113 Beta2AR antagonists increasekeratinocyte migration by preventing the bindingof endogenously or systemically synthesizedepinephrine.104,113 These studies have clinical im-plications, providing a framework for understand-ing how chronic stress impairs healing and atherapeutic target for improving healing. Indeed,anecdotal case reports support the use of beta ad-renergic antagonists as therapeutic agents to healchronic wounds.120 The unique ability of the skin towork as an independent neuroendocrine system al-lows for both local and systemic hormone-mediatedresponses in homeostasis and wound healing.

Keratinocyte immunityduring epithelialization

A well-executed immune response is also es-sential for successful wound healing. The skin has

Table 1. Epidermis is a neuroendocrine organ

Ligand Receptor Epidermal Layer Function Reference

Corticotropin-releasinghormone (CRH)

CRH-R1 Present throughout epidermis Inhibits keratinocyte proliferation 112,176–178

Enhances the production and secretion of the POMC peptidesin keratinocytes, melanocytes, endothelial cells, andcutaneous nerves

Stimulates production of steroids in the skin

Class II PTH/PTHrP Class II PTH/PTHrP-R Highly expressed between basalto granular layers

Inhibits keratinocyte proliferation and stimulatesdifferentiation

108,112

Restoration of epidermal homeostasis during wound healing

Vitamin D Vitamin D-R All epidermal layers exceptstratum corneum

Inhibits keratinocyte proliferation and stimulates keratinocytedifferentiation

112,179

Testosterone dihydro-testosterone

Androgen-R Restricted to basal layer Inhibits wound healing, promotes inflammation, regulateskeratinocyte proliferation and differentiation, and reducespermeability barrier function of stratum corneum

180–182

In genital skin present in spi-nous and granular layers

Androgen receptor antagonist: accelerates wound healing

Estrogen Estrogen-R (nuclearERb)

ERb: present in the spinous andbasal layers

Stimulates keratinocyte proliferation and acceleratesepithelialization

117,178,182,183

Decreases inflammatory response and suppresses apoptosis

Glucocorticoid Glucocorticoid-R Present throughout theepidermis, but mainlyexpressed in basal layer

Inhibits epithelialization 107,179,181

Represses expression of basal cell-specific keratins K5, K14,and wound healing-associated keratins K6, K16, K17

Prolonged exposure can defect the permeability barrierfunction

Mineralocorticoid Mineralocorticoid-R Present throughout epidermis Aldosterone/MR affect sodium, potassium, or calciumcurrents in keratinocytes, leading to altered epidermal ionhomeostasis

178,184

Acetylcholine Muscarinic-R Predominantly in the basal orsuprabasal layers

Regulate keratinocyte proliferation, migration, anddifferentiation

112,185,186

Acetylcholine Nicotinic-R Throughout epidermis, but pre-dominantly in the basal orsuprabasal layers

Stimulates keratinocyte motility and differentiation 112,185

The receptors represent functional ion channels mediatingthe influx of sodium and calcium, and the efflux ofpotassium, being essential for keratinocyte viability

ACTH Adrenoreceptors MC1Rand MC2R

MC1R: strongly expressed insuprabasal layer; MC2R:present throughout epidermis

Stimulates de novo cortisol synthesis in keratinocytes 112,187–189

Regulates keratinocyte differentiation

POMC, proopiomelanocortin.


traditionally been regarded for its protective role asa physical barrier to foreign pathogens. Morerecently, however, several resident skin cell popu-lations, including keratinocytes, have gained rec-ognition for their active contribution to hostimmune responses during epithelialization.

Keratinocytes perform functions that directlyinfluence both immunity and wound closure.Although wound re-epithelialization is driven pri-marily by keratinocyte migration and prolifera-tion, in this process, keratinocytes simultaneouslyemploy strategies to resist microbial entry. Kera-tinocytes recognize foreign pathogens and endog-enous danger signals through their Toll-likereceptors (TLRs), specifically, TLRs 1–6 and 9.121–123

TLR activation initiates effector immune cellrecruitment, through the production of proin-flammatory cytokines and chemokines, and hasrecently been implicated in the process of woundclosure and re-epithelialization.124,125 In particu-lar, TLRs 2, 3, 4, and 9 have been identified as keymodulators of wound epithelialization. The in-creased expressions of TLR2 and TLR4 have beenshown to contribute to impaired wound healing inanimal models.125,126 Furthermore, an associationbetween nonhealing chronic venous ulcers andchronic inflammatory cytokine expression second-ary to persistent activation of TLR2 and TLR4 hasbeen described.127 More recently, TLR4 was shownto promote optimal early inflammation in woundrepair. Mice deficient in TLR4 experienced signif-icantly delayed wound closure as well as impairedinflammatory cell infiltration.128 Similarly, TLR3,a recognized initiator of inflammation followinginjury,129 and TLR9, have been reported to accel-erate wound closure and epithelialization.130,131

The discrepancies in the above findings underscorethe need to maintain a delicate balance and tem-poral regulation of TLR activation to return theskin to homeostasis following acute injury.124 Onesuch way to harness TLR-induced inflammationmay involve commensal skin bacteria.129

Following wounding and/or microbial challenge,keratinocytes are also stimulated to release anti-microbial peptides (AMPs), including humancathelicidins, hCAP 18 and LL-37, and humanb-defensins.132 Ubiquitous in nature, AMPs arepotent antimicrobial agents that are effectiveagainst both gram-positive and gram-negativeorganisms. These proteins have been shown to becritical in wound closure and epithelialization.Levels of AMPs increase significantly in earlywound healing and subsequently decrease uponwound closure.133,134 However, expression of AMPsis deregulated in chronic wounds; keratinocytes in

nonhealing wounds show a constitutively highbaseline expression of human b-defensin-2 (hbD-2)and psoriasin, while expression of RNase 7 andLL-37 is suppressed.133,135 Nevertheless, AMPsare still considered as potential antimicrobialtherapeutics for chronic wounds; as an example,topical LL-37 has been reported to accelerate epi-thelialization in acute wounds in murine mod-els.136 In the porcine wound model, the cathelicidinprotein PR-39 was reported to induce syndecans inacute wounds.137 Syndecans have been suggestedto play a key role in appropriate wound healing, asmice deficient in syndecan-4 genes were observedto have delayed wound closure.138 Most recently,the critical role of regenerating islet-derivedprotein 3-alpha (REG3A) and regenerating islet-derived protein 3 gamma (RegIIIc) in wound epi-thelialization was described.139 Reg3A and RegIIIchave been shown to function as antimicrobialproteins in the intestine, inhibiting microbialproliferation.140 These proteins were also found toinhibit keratinocyte differentiation/proliferation,thus facilitating wound closure.139

In response to various insults and injuries,keratinocytes can also activate inflammasomes.141

Inflammasomes are multiprotein innate immunecomplexes that produce and mature proinfla-mmatory cytokines upon recognition of foreignpathogens. The role of inflammasomes in woundhealing has recently been explored. Levels of epi-dermal caspase 8, a mediator of apoptosis, fluctuategreatly during the course of wound repair. The loss ofepidermal caspase 8 led to the induction of aberrantcross talk among epithelial, mesenchymal, and leu-kocytic cells. This ultimately resulted in epidermalhyperplasia and the alteration of the wound repairprocess, highlighting the need for an appropriateinflammasome response for wound closure.142

The immunologic function of the skin reliesheavily on interactions among keratinocytes andother immune cells. Keratinocytes are the mainprotagonists of skin innate immunity, which fur-ther control the adaptive immune response duringepithelialization and wound closure. Keratinocytesclearly participate in host defense against invadingmicroorganisms in multiple ways during normaland impaired wound healing, and one approachto understanding the skin–bacteria interactionduring wound closure includes recently advancedmicrobiome studies.

Skin microbiome and wound healingA microbiome can be defined as the entirety of all

microbes, their genomes, and interactions within aparticular environment. Recent advances in high-

456 PASTAR ET AL.

throughput sequencing technologies have facili-tated studies of the complex microbial inhabitantsof the human body by providing a more compre-hensive identification of microbes than traditionalculture methods.143 These novel methods use 16SrRNA gene-based analyses to characterize the di-versity of microbial communities present at specifictissues or body locations, including skin.143 Al-though the complexity and diversity of healthyhuman skin microflora have been established,144 amore thorough understanding of skin microbialinhabitants and pathogens can provide a founda-tion for future research regarding the interactionsbetween microbes and epidermis, during normal,acute, and impaired wound healing. As the epi-dermis is the first line of host defense against theexternal environment, defining wound micro-biomes conducive to the normal wound healingprocess is important and may greatly benefit pa-tients with nonhealing wounds. The power of the16s rRNA sequencing technology of microbiomes isshown by a recent study indicating a shift in bothunwounded skin and wound microbiota in diabeticmice (db/db) that was used as a model of impairedwound healing.145 Forty times more bacteria anddifferent types were found on the skin of db/db miceand impaired healing coincided with a shift in mi-crobial diversity, which changed over time duringhealing.145 The human skin microbiome undergoesdynamic changes after barrier disruption as well.The most recent study suggested that the micro-biome of the deeper stratum corneum layers playsan important role in the microbial re-colonizationprocess of the human skin after injury caused bytape stripping.146 It has also been suggested thatmicrobiomes of chronic wounds play significantroles in impaired wound healing, in the presence orabsence of clinical signs of infection.147–150 Thispolymicrobial bioburden in chronic wounds existspredominantly in the form of a biofilm,151 whichcan inhibit keratinocyte migration and epithelial-ization.152,153 The use of standard clinical micro-bial cultures to diagnose the wound bioburden hasbeen shown to be of limited value as culture tech-niques typically detect only microorganisms thatgrow relatively quickly and easily in laboratorymedia,154 while 16S rRNA gene-based analyseshave revealed more complex wound microbiomesthan those described previously in culture-basedanalyses.147–149 It has also been suggested that thediversity of the chronic wound microbiome is sig-nificantly reduced compared with neighboringhealthy skin.148 Depending on the study and thetype of chronic wounds evaluated, among the mostprevalent genus in chronic wounds were Staphylo-

coccus, Pseudomonas, and Corynobacterium, whileobligate anaerobes Bacteroides, Peptoniphilus,Fingoldia, Anaerococcus, and Peptostreptococcusspp were identified as well.148,150 In addition, theprevalence of Oxalobacteraceae, Corynebacter-iaceae, and Pseudomonadaceae was increased bythe use of antibiotics.149 Recent studies using ani-mal models of wound infections have shown thatpolymicrobial infection with S. aureus and Pseudo-monas aeruginosa inhibits epithelialization signifi-cantly more than infection with single species,153

suggesting that the complex chronic wound micro-biome may have even more of a detrimental effect onwound closure.

Despite these molecular advances, diagnosis ofwound infection in the clinical setting is still basedon standard microbiology culturing techniques,making detection of biofilm and anaerobic speciesimpossible. A better understanding of the bacterialpopulations associated with clinical outcomes isessential to improve the management of chronicwounds. In the future, we can expect the develop-ment of quantitative PCR-based methods that willenable more detailed analyses of target taxons ofthe human cutaneous and wound microbiota,complementing high-throughput sequencing andenabling detection of wound pathogens that wouldinfluence treatment decisions to benefit patientswith chronic nonhealing wounds.

Models to study epithelializationTo assess keratinocyte migration in response to

wounding and specific treatments (e.g., pharma-cological agents), the scientific research commu-nity employs a range of models using keratinocytecultures, cultured fetal, adult, or engineered hu-man skin, as well as various animal models(Table 2).107,155–158 Due to space limitation, we willdescribe only the most frequently utilized woundhealing models. It should be kept in mind that theetiology of human chronic wounds often includes acombination of multiple factors and underlyingcomorbidities, and that various models typicallyaddress a single factor (e.g., diabetes, infection, andischemia). In spite of research efforts focused onimproving existing models and developing newones, a model that fully replicates human non-healing chronic wounds remains missing.

A widely used in vitro assay to study epithelial-ization is a wound scratch assay, where humankeratinocytes are grown to confluence in a tissueculture dish and then wounded by a scratch using apipette tip.40,61,159 As the scratch wound gap closes,keratinocyte migration is microscopically recordedand quantified by image analysis. This model al-


lows for quantifying only migration, by inhibitingproliferation using, for example, mitomycin Cpretreatment. As a caveat, however, data obtainedusing this technique should be corroborated usingother model systems since in vitro conditions can-not mimic the complexity of cross talk betweendifferent cell types that occurs during the woundhealing process in vivo.

However, there are obvious difficulties in sam-pling skin acute wounds in humans due to ethicalconcerns. To overcome this obstacle, a first reportmodeling the wound healing process in humanskin using organotypic cultured skin was reportedby Kratz.160 In this model, both epidermal anddermal cells maintained their viability in incisionalwounds created in ex vivo cultured skin, maintainedfor up to 14 days in either 2% or 10% fetal bovineserum (FBS). Skin explants maintained in low FBSdid not show signs of epithelialization, whereaswounds maintained in high FBS showed completeepithelialization after 14 days.160 Our laboratoriesdeveloped an ex vivo acute wound model utilizing

human skin obtained from reduction surgeries inwhich we create acute wounds using a biopsy punch,maintaining them at the air–liquid interface to as-sess the epithelialization process.85,107,115,155,161

Details of the wounding technique can be found inthe cited publications.161,162 Recently, two new exvivo wound models, a partial-thickness human skinculture model,163 and linear excisional woundmodel164 were described, further confirming thevalidity and usefulness of ex vivo human woundmodels. Having in mind the limitation of materialthat can be obtained from cosmetic surgeries,bioengineered skin substitutes offer the advantageof creating multiple wounds from the same tis-sue source, minimizing experimental variability.These bioengineered skin substitutes use keratino-cytes grown on fibroblast feeder layers thus reca-pitulating skin morphology.

Advances in tissue engineering have facilitatedthe progress of the development of diverse modelsto study wound re-epithelialization and providedour research community with both murine and

Table 2. Summary of frequently used models to study wound epithelialization

Advantage Disadvantage Reference

In vitroWound scratch assay Utilizes human epidermal keratinocytes

By inhibiting proliferation, one can only assesskeratinocyte migration and its underlyingbiology

Lack of cross talk with other cell typesinvolved in the wound healing process

40,61,159

Ex vivoHuman ex vivo acute wound model Presence of full-thickness epidermis and dermis;

Langerhans cells, pigment cells, and nerveendings

Absence of immune cells and blood supplyVariation in wound depth

85,107,115,155,161,162

Human ex vivo linear excisionalwound model

Standardized wound depthPresence of full-thickness epidermis and dermis;

langerhans cells, pigment cells, and nerveendings present

Absence of immune cells and blood supply 164

Partial-thickness human skin culturemodel (incision)

Presence of full-thickness epidermis and dermis;langerhans cells, pigment cells, and nerveendings present

Absence of immune cells and blood supply 163

Murine and human skin equivalents Ability for studying the interaction betweenkeratinocytes and fibroblasts

Lack of immune cells and blood supply;simplified ECM

157,165

In vivoTape stripping Partial removal of epidermis

Several levels of barrier disruption can beinduced

Variation in depth of barrier disruption 190

Murine incisinal/excisionalwound model

Ability for studying the interaction and influenceof different cell types on epithelialization

Use of wound splinting in full-thickness woundsto minimize contraction

Uncomplicated standardization and multiplereplicates

Rodents heal by contraction of the subcu-taneous muscle

Variation in skin anatomy and physiologyin rodents vs. humans

Influence of the hair follicle phase onepithelialization

77,85,158,167,170,191

Porcine partial/full-thicknesswound model

Greatest structural skin similarity and the woundhealing process to the humans

Multiple replicates within single animal

Lack of commercially available reagents foranalyses

107,172,192,193

Rabbit ear wound model Decreased blood flowAbsent wound contractionMultiple replicates within single animal

Dermis firmly attached to underlyingcartilage

Avascular wound base

171,194

ECM, extracellular matrix.

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human skin equivalents.157,165 Benefitssuch as uncomplicated standardization, apossibility of multiple repetitions, as wellas following a wound progression throughtime are advantages that have promotedwide utilization of animal models forwound healing studies. The Drosophilawound model provided insight on how thecytoskeletal machinery is involved in theepithelialization process.166 Further-more, small mammals such as mice andrats allow scientists for genetic manipu-lation, utilizing overexpression orknockout technologies to specifically tar-get keratinocytes and focus on the epi-thelialization process, or by mimickingdiseases known to affect the woundhealing process such as diabetes, dysli-pidemia, and obesity.77,85,156 In studiesutilizing small animals, usually a woundis created on the dorsum of the animal,by tape stripping, incisional or excisionalwounding, and the rate of wound epi-thelialization is measured using histo-morphometric analysis.77,85,158 One ofthe widely adopted full-thickness mousemodels utilizes wound splinting. This modelminimizes contraction, a mechanism by whichmurine wounds mostly heal, and allows woundhealing to occur primarily through the processes ofgranulation and re-epithelialization.158,167 How-ever, differences in skin composition (e.g., thick-ness of skin and number of HFs per cm2) should beconsidered when obtained data are translated tothe human skin. To address these dissimilarities, amodel where full-thickness human skin is trans-planted onto nude animals has been devel-oped.168,169 In addition, recent work by Ansell et al.described the influence of the different hair-cyclestages on murine skin wound healing. The authorsshowed that murine skin wound healing is accel-erated during the anagen phase of HF cycling, thusraising caution in regard to interpretation of ob-tained data when murine wound healing studies areemployed.170 A rabbit ear model is also used to as-sess re-epithelialization and limits wound contrac-tion by virtue of the underlying cartilage splintingthe wound.171 The porcine wound model, however,either with partial- or full-thickness skin wounds,has been shown to have the greatest similarity tothe human wound healing process.107,172 Porcineskin is structurally most similar to human skin,including parameters such as epidermal thicknessand dermal–epidermal thickness ratios, as well assimilar patterns of HFs and blood vessels.173 Por-

cine wounds heal primarily by epithelialization,whereas murine, rabbit, and dog wounds heal pri-marily by contraction.174 Epithelialization in a por-cine wound healing model can be also assessed usingthe salt-split technique in conjunction with histo-logical evaluation.175 Taken together, wound heal-ing studies conducted on animals are extremelyvaluable and allow for initial preclinical evaluationfor testing potential treatment modalities.

AUTHOR DISCLOSUREAND GHOSTWRITING STATEMENT

No competing financial interests exist. The con-tent of this article was expressly written by theauthors listed.

ABOUT THE AUTHORS

Irena Pastar, PhD, holds a faculty position asan Assistant Professor at the Department of Der-matology and Cutaneous Surgery, University ofMiami Miller School of Medicine. Olivera Stoja-dinovic, MD, is a Research Assistant Professor atthe Department of Dermatology and CutaneousSurgery, University of Miami Miller School ofMedicine. Horacio Ramirez, Andrew Sawaya,Natalie C. Yin, Shailee B. Patel, Aron G. Nus-baum, and Laiqua Khalid are graduate studentsand medical fellows at the University of Miami

TAKE-HOME MESSAGES� The epidermis is a stratified epithelium comprising several layers of

keratinocytes.

� Keratinocytes are responsible for restoring the epidermis after injurythrough a process known as epithelialization.

� Keratinocytes continually renew during homeostasis as well as duringdisruption of the epidermal barrier.

� The regenerative capacity of the skin relies on the local populations ofESCs.

� Multiple regulators such as growth factors and cytokines, integrins,keratins, MMPs, chemokines, and extracellular matrices modulate ker-atinocyte migration during wound closure.

� The epithelialization process is impaired in all types of chronic wounds.

� Novel mechanisms of gene expression regulation, including miRNA andhistone modifications, are important for re-epithelialization.

� Keratinocytes produce various hormones, neuropeptides, and neuro-transmitters, and express their receptors that help in maintaining epi-dermal homeostasis and are implicated to have a role in wound closure.

� Keratinocytes actively participate in host defense against invading mi-croorganisms during wound healing.

� A better understanding of the epithelialization process may ultimatelylead to the development of novel therapeutic approaches for nonhealingwounds.


Miller School of Medicine. Rivkah R. Isseroff,MD, is Professor of Dermatology at the UC Davis,with the strong research and clinical interest inwound care. Marjana Tomic-Canic, PhD, is aProfessor of Dermatology, and Director of theWound Healing and Regenerative Medicine Re-

search Program at the Department of Dermatologyand Cutaneous Surgery, University of MiamiMiller School of Medicine. Dr. Tomic’s researchfocuses on molecular and cellular mechanisms oftissue repair and regeneration in skin and itspathogenesis.

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Abbreviations and Acronyms

11bHSD1¼ 11b-hydroxysteroiddehydrogenase 1

ACTH¼ adrenocorticotropic hormoneADAM12¼ disintegrin and

metalloprotease 12AMP¼ antimicrobial peptides

beta2AR¼ beta2-adrenergic receptorBM¼ basement membrane

CRH¼ corticotropin-releasing hormoneCYP11B1¼ steroid 11 b-hydroxylase

DP¼ dermal papillaECM¼ extracellular matrixECs¼ endothelial cellsEGF¼ epidermal growth factor

EGR3¼ early growth response factor 3ESCs¼ epidermal stem cellsEzh2¼ enhancer of zeste homolog 2FBS¼ fetal bovine serumFGF¼ fibroblast growth factorsFPP¼ farnesyl pyrophosphateGCs¼ glucocorticoids

GM-CSF¼ granulocyte–macrophagecolony-stimulating factor

H¼ histoneH3K27me3¼ histone H3 lysine 27

trimethylationHF¼ hair follicle

hbD-2¼b-defensin-2 and psoriasinIFE¼ interfollicular epidermisIGF¼ insulin-like growth factorIRS¼ inner root sheet

K¼ keratinLepR¼ leptin receptorLN5¼ laminin-5

MCP-1¼macrophage chemoattractantprotein

miRNAs¼microRNAsMMPs¼matrix metalloproteinases

ORS¼ outer root sheetPCR2¼ polycomb repressive complex 2

PKC¼ protein kinase CPOMC¼ proopiomelanocortinPTHrP¼ parathyroid hormone-related

protein proteinREG3A¼ regenerating islet-derived protein

3-alphaRegIIIc¼ regenerating islet-derived protein

3 gammaRISC¼ RNA-induced silencing complex

SG¼ sebaceous glandTIMPs¼ inhibitors of metalloproteinase

TLRs¼ Toll-like receptorsTNF¼ tumor necrosis factor

VEGF¼ vascular endothelial growthfactor

464 PASTAR ET AL.

Epithelialization in Wound Healing: A Comprehensive Review

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