a review of the influence of growth factors and cytokines in in vitro human keratinocyte migration

Cytokine 62 (2013) 1–21

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Cytokine

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Review Article

A review of the influence of growth factors and cytokines in in vitro humankeratinocyte migration

Philip V. Peplow a,⇑, Marissa P. Chatterjee b

a Department of Anatomy, University of Otago, New Zealandb Department of Prosthetic Dentistry, BIOMAT Research Cluster, KU Leuven, Belgium

a r t i c l e i n f o a b s t r a c t

Article history:Received 5 November 2012Received in revised form 4 February 2013Accepted 11 February 2013Available online 11 March 2013

Keywords:Growth factorCytokineKeratinocyteCell migrationIn vitro

1043-4666/$ - see front matter � 2013 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.cyto.2013.02.015

⇑ Corresponding author. Tel.: +64 3 479 7374; fax:E-mail address: [email protected]

Objective: Keratinocyte migration from the wound edge is a crucial step in the reepithelization of cuta-neous wounds. Growth factors and cytokines, released from cells that invade the wound matrix, play animportant role, and several in vitro assays have been performed to elucidate this. The purposes of thisstudy were to review in vitro human studies on keratinocyte migration to identify those growth factorsor cytokines that stimulate keratinocyte migration and whether these assays might serve as a screeningprocedure prior to testing combinations of growth factors or cytokines to promote wound closure in vivo.Methods: Research papers investigating effect of growth factors and cytokines on human keratinocytemigration in vitro were retrieved from library sources, PubMed databases, reference lists of papers, andsearches of relevant journals.Results: Fourteen different growth factors and cytokines enhanced migration in scratch wound assay andHGF together with TGF-b, and IGF-1 with EGF, were more stimulatory than either growth factor alone.HGF with TGF-b1 had a greater chemokinetic effect than either growth factor alone in transmigrationassay. TGF-b1, FGF-7, FGF-2 and AGF were chemotactic to keratinocytes. EGF, TGF-a, IL-1a, IGF and MGSAenhanced cell migration on ECM proteins.Conclusion: Many growth factors and cytokines enhanced migration of keratinocytes in vitro, and certaincombinations of growth factors were more stimulatory than either alone. These and other combinationsthat stimulate keratinocyte migration in vitro should be tested for effect on wound closure and repairin vivo. The scratch wound assay provides a useful, inexpensive and easy-to-perform screening methodfor testing individual or combinations of growth factors or cytokines, or growth factors combined withother modalities such as laser irradiation, prior to performing wound healing studies with laboratoryanimals.

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1. Introduction

In the repair of cutaneous wounds it is essential for the wound tobe resurfaced as quickly as possible in order to regain structural andfunctional integrity and to serve as a protective barrier to preventdesiccation and invasion by pathogenic microorganisms. This isachieved by proliferation and directional migration of keratinocytesfrom the edges of the wound. For migration to occur there needs tobe disruption of intercellular junctional complexes. The migrationof keratinocytes occurs initially over a provisional matrix thatforms within and fills the wound bed. Fibrin, fibronectin and vitro-nectin together with platelets are components of this matrix [1] andit provides a scaffold or mesh for neutrophil, macrophage, lympho-cyte, endothelial cell, and fibroblast localization at the wound site.Fibrinogen and fibronectin when covalently linked by factor XIII of

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the blood coagulation system promote the adherence of fibroblaststo tissue culture dishes [2] and fibronectin coating of plates in-creases the adherence of endothelial cells [3,4]. As wound repairprogresses, the provisional matrix becomes replaced by one con-sisting of collagen fibers synthesized by fibroblasts, and of proteo-glycans such as hyaluronic acid, heparan sulfate, chondroitinsulfate, dermatan sulfate and which contribute to the extracellularmatrix (ECM). The wound healing process is orchestrated by growthfactors and cytokines released by a variety of cells that accumulatewithin the provisional matrix and ECM (e.g. platelets, neutrophils,fibroblasts, endothelial cells, macrophages, lymphocytes).

The ECM can directly bind to and release certain growth factors(e.g. heparan sulfate binding to fibroblast growth factor-2, FGF-2)which may serve to sequester and protect growth factors from deg-radation and/or enhance their activity. Indirect interactions in-clude binding of cells to ECM via integrins, which enables cells torespond to growth factors (e.g. integrin binding is necessary forvascular endothelial growth factor (VEGF)-induced angiogenesis)and can induce growth factor expression (e.g. adherence of

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2 P.V. Peplow, M.P. Chatterjee / Cytokine 62 (2013) 1–21

monocytes to ECM stimulates the synthesis of platelet-derivedgrowth factor, PDGF) [5].

Tight junctions are considered to be crucial for the barrier func-tion of the mammalian skin and have been shown to be present inthe epidermis of wild-type mice [6], and immunofluorescencelabeling and immunoelectron microscopy with antibodies to ZO-1and occludin demonstrated the presence of tight junction antigensin adult and developing human epidermis [7]. Interkeratinocyteadherens junctions have also been shown in human epidermis[8]. Release from tight and adherens junctions causes the loss ofepithelial cell polarity and enables migration of keratinocytes.Adult epidermal keratinocytes migrate by crawling, a process thatrequires protrusion of the plasma membrane at the front of the celland contraction of the cell body at the rear [9]. Lamellipodia aresheet-like extensions of cytoplasm that form transient adhesionswith the cell substrate, enabling the cell to move along the sub-strate. Formation of lamellipodia requires ruffling along the plasmamembrane and reorganization of the actin cytoskeleton [10,11].Various growth factors can influence keratinocyte shape differ-ently. For example, insulin-like growth factor-1 (IGF-1) stimulatesmembrane protrusion and facilitates cell spreading, while epider-mal growth factor (EGF) induces contraction of keratinocytes [12].The effects of each growth factor on keratinocyte shape are medi-ated by distinct signal transduction pathways.

Growth factors and cytokines play an important role in thewound healing process. They induce proliferation and migrationof cells within the wound environment, and also regulate theECM by stimulating the synthesis of collagen and fibronectin in avariety of cell lines [13] as well as many other ECM components[14]. Keratinocyte proliferation and migration are essential forthe formation of skin appendages and epidermal repair afterwounding, and many growth factors investigated so far have beenshown to stimulate these functions [15]. The question arises as towhether there are specific contributions of individual growth fac-tors and cytokines to particular keratinocyte functions. This reviewhas searched published in vitro human studies and analyzed andcompared various growth factors and cytokines for their potentialto regulate the shape and spreading of individual keratinocytes,stimulate migration in scratch wound assay and in Boyden andtranswell chambers, and to influence migration with specificECM proteoglycans as substrate.

2. Materials and methods

2.1. Literature review

A systematic review of the relevant literature was performed.Original research papers investigating the effects of growth factorsand cytokines on the shape, spreading and migration of humankeratinocytes in vitro, and published up to December 2011 wereretrieved and used for this review. Relevant papers were soughtand obtained from library sources and the online database PubMedusing EndNote X1 (Thomson Reuters, Carlsbad, USA) or the webdatabase accessed at www. pubmed.com. Search terms were‘‘keratinocyte’’, ‘‘growth factor’’, ‘‘cytokine’’, ‘‘chemokine’’, ‘‘effectof growth factor’’, ‘‘effect of cytokine’’, ‘‘effect of chemokine’’,‘‘scratch wound’’, ‘‘migration’’, and ‘‘in vitro’’. Additional secondarysources of information included reference lists from retrieved pa-pers, and pertinent papers identified by hand searches of relevantjournals not found from the databases.

2.2. Inclusion/exclusion criteria

We included studies that met the following c riteria: (i) growthfactor or cytokine was investigated as the primary intervention

(independent variable); (ii) the growth factor or cytokine togetherwith dose and exposure time were defined; (iii) at least one out-come or index of cellular expression of shape or migration wasidentified; (iv) studies were performed with human keratinocytecells or cell lines in vitro; (v) studies were related to epidermalregeneration and wound repair.

Studies excluded from this study were: (i) in vitro studiesinvolving keratinocytes from animals other than humans; (ii)in vivo studies involving human patients or whole animals, not iso-lated keratinocyte cells or cell lines; (iii) studies performed withcells other than keratinocytes; (iv) reviews and meta-analyses;(v) studies for which only an abstract was available; (vi) studies re-ported in languages for which no English language translation wasavailable; (vii) in vitro studies which did not examine the effect ofgrowth factors or cytokines on keratinocyte migration.

2.3. Retrieved articles

The process followed for retrieving articles from the literaturesearch is summarized in Fig. 1.

2.4. Items for data extraction

Literature searches were carried out by both authors and thefindings were combined. Articles for inclusion and exclusion wereidentified independently, and confirmed, thereby minimizing bias.

For included articles, the following data were extracted and tab-ulated: research method: (in vitro studies – controls, minimizingvariability in experimental conditions); cell type: (human keratino-cyte cells; donor tissue source or established cell lines); descriptionof cells: (normal cells, cell lines, number of replicates); growth factoror cytokine exposure parameters; experimental outcomes: (cell shape,cell spreading, migration); study conclusion: (results of exposure ofkeratinocytes to growth factor or cytokine).

Studies were then critically reviewed in terms of methodology,appropriateness of growth factor and cytokine parameters, andcontributions of individual growth factors and cytokines to partic-ular keratinocyte functions.

3. Results

Results from the literature search are summarized in Fig. 1. Intotal, 24 publications were included in this review [12,16–38]and are summarized in Tables 1–6. Several of the publicationshad performed more than one type of study (e.g. scratch wound as-say and transmigration assay using chambers).

3.1. Migration of keratinocytes in scratch wound assay or similarprocedure (Tables 1 and 2)

There were 11 studies in which the cell monolayer waswounded with a pipette tip or spatula, two studies where half ofthe monolayer was removed with a razor blade, and one studywhere cell monolayer was denuded by a rubber policeman. Sevenof the studies had used HaCaT cell line, one with NHEK cell line,and six using keratinocytes grown from donor tissue. All of thestudies had used cells cultured to complete or almost completeconfluence, and in five of the studies the cells were incubated withmitomycin C to arrest proliferation prior to wounding. The cellswere starved prior to scratch wounding or removing part of themonolayer, and the extent of migration of cells into the scratchwound area or the area cleared of cells determined at times thatvaried from 4 to 72 h (mean 31 h). With prior starvation and with-out mitomycin treatment or added growth factor or cytokine,keratinocyte migration achieved only a very small extent of wound

Fig. 1. Flowchart of Literature Search: Studies of influence of growth factors and cytokines on migration of human keratinocytes in vitro.

P.V. Peplow, M.P. Chatterjee / Cytokine 62 (2013) 1–21 3

closure (5%), thereby demonstrating a quiescent state ofkeratinocytes.

The influence of EGF was examined in six of the studies and atall of the concentrations tested that ranged from 0.1 to 100 ng/ml,it increased the migration of keratinocytes. Interestingly it stimu-lated migration in the presence of both low (0.09 mM) and highCa2+ (1.2 mM). Heparin binding EGF (HB-EGF) at 1 ng/ml stimu-lated migration. Transforming growth factor-a (TGF-a), which be-longs to the EGF family of growth factors, was tested in four studiesat 3, 6 and 55 ng/ml and increased keratinocyte migration at eachof these concentrations. TGF-a caused a greater increase in kerati-nocyte migration than EGF when both growth factors were testedat 10 nM concentration (170 and 64 ng/ml, respectively).

Transforming growth factor-b1 (TGF-b1) was investigated infour of the studies and this growth factor at 2 and 10 ng/ml en-hanced keratinocyte migration, but at 25 ng/ml it suppressedmigration.

Keratinocyte migration in the presence of hepatocyte growthfactor (HGF) was examined in three studies. One study showed

HGF at 6 ng/ml to be effective in stimulating migration. In one ofthe other studies, HGF at 25 ng/ml resulted in only a very small in-crease in wound closure (7%), whereas wound closure with HGF at100 and 250 ng/ml was 52% and 62%, respectively. In the thirdstudy, HGF at 54 ng/ml enhanced cell migration. HGF at 6 ng/ml to-gether with TGF-b1 at 2 ng/ml was more effective than eithergrowth factor alone in stimulating keratinocyte migration.

Members of the fibroblast growth factor family were tested infour of the studies. In one study, each of FGF-10 (also known askeratinocyte growth factor-2, KGF-2) and FGF-7 (KGF-1) at 10 ng/ml had no effect on the migration of NHEK cells, but FGF-10 incombination with soluble dermatan sulfate (DS) 2 lM stimulatedmigration of NHEK cells. FGF-7 in combination with DS 2 lM didnot significantly increase migration of these cells. In the otherstudies which used keratinocytes or HaCaT cell line, FGF-7 at 2and 19 ng/ml enhanced cell migration. FGF-2 (basic FGF) wastested in two studies at 22 ng/ml and stimulated keratinocytemigration. FGF-7 was as potent as TGF-a and both moreactive than FGF-2 when all were tested at 1 nM concentration

Table 1Migration of keratinocytes in scratch wound assay or similar procedure.

Authors,reference

Type of cells Culture medium and plating density of cells Cell culture Growth factor or cytokineadded to culture medium,and duration of treatmentof cells with growth factoror cytokine

Experimental outcomes and time whenmeasured after growth factor orcytokine treatment

Tochioet al.[16]

HumankeratinocytesHaCaT cell line

Keratinocytes were grown in DMEM with 10% FBS.Approximately confluent cells were starved for 24 h inDMEM containing 0.1% FBS and then treated with EGF.After a standardized cell-free (wound) area was made byscraping the culture surface using a plastic pipette tip,cells were incubated for 8 h in DMEM containing 10%FBS or 0.1% FBS with EGF. Phase-contrast images takenimmediately and 8 h after scraping were captured in 8–10 fields per well

Change in wound area for 8 h indicates wound recoveryarea

rEGF (0.1, 0.5 and 1 ng/ml = 0.016, 0.08 and0.16 nM)

Migration assessed based on the meanvalue of wound recovery area per fieldin each well at 8 h

Cells were seeded (5 � 104/well) in culture plates or oncoverslips, cultured to 90% confluence in DMEM with10% FBS, transfected with 12 pmol siRNAs for 8 h.Knockdown of ALDOA mRNA by siRNA transfection withits siRNAs was examined by qRT-PCR and Western blotanalysis

Number of lamellipodia-forming cells in the edge ofwound recovery area counted and divided by number oftotal cells in edge of wound recovery area. This valuewas designated as rate of lamellipodia-forming cells

8 h Rate of lamellipodia-forming cells ineach experiment was calculated bycounting at least >50 cells with orwithout lamellipodia in a tip region of awound recovery area

Comments on study design and findingsWound recovery area in presence of 0.1, 0.5 and 1 ng/ml EGF was significantly increased compared with control. Simultaneously ALDOA mRNA and protein levels were increased by EGF treatment. Knockdown of ALDOA

(fructose-1,6-biphosphate aldolase A) suppressed lamellipodia formation and migration of keratinocytes

RasanenandVaheri[17]


Keratinocytes were cultured in DMEM with 5% FBS To confirm that TGF-b1 causes proliferation arrest,keratinocytes were seeded in 96-well plates(2 � 104 cells/well) and let to plate for 2 h. Culturemedium with unattached cells was aspirated andreplaced with 100 ll of either control medium or TGF-b1. Cells were incubated for 48 h after which MTTreagent (5 mg/ml) was added and incubated for 4 h.Culture medium was aspirated and 100 ll of crystaldissolving solution was added to each well andabsorbance measured at 540 nm

rhTGF-b1 (2 ng/ml = 0.08 nM)

Migration of cells measured at 24 hfollowing wounding by a scratch

For scratch-wound assay, cells were grown toconfluence, serum-starved and scratched with pipettetip to create a wound. After wounding, cells werewashed twice with serum-free medium to remove celldebris and grown for 24 h in control or TGF-b1-containing medium. Photographs were taken andpercentage of the open wound area at 24 h was analyzed

Levels of epithelial junction proteins ZO-1 and E-cadherin in cells in response to TGF-b1 wereinvestigated by immunoblotting andimmunofluorescence

Migration of cells: 24 h Proliferation of cells measured by MTTassay at 48 h. Levels of epithelialjunction proteins at 48 h measured byimmunoblotting andimmunofluorescence

Proliferation assay: 48 hLevels of epithelial junctionproteins: 48 h

TGF-b1 caused proliferation arrest of keratinocytes. Expression of ZO-1 decreased in response to TGF-b1 treatment. E-cadherin level was downregulated to a lesser extentIn scratch-wound assay, wound closure in TGF-b1-treated cells was significantly increased

Nam et al.[18]


Keratinocytes were grown in RPMI-1640 medium with10% FBS

rhHGF (30units/ml � 6 ng/ml = 0.1 nM)

Motility of cells determined bymeasuring width of wound in scratch-wound assayFor scratch-wound assay, cells were seeded

(3 � 105 cells/well) into 24-well plates in RPMI-1640with 10% FBS and allowed to adhere overnight. Cellswere then starved with 0.5% FBS containing medium.The monolayers were carefully scratched with sterilepipette tips and incubated with 0.5% FBS containingmedium with HGF and/or TGF-b1. After 36 h, cells werewashed 3� with cold PBS, fixed in acetone:methanol(1:1 v/v) for 15 min on ice, and stained with 2% (w/v)crystal violet. The width of the wound was measured

rhTGF-b1 (2 ng/ml = 0.08 nM) 36 h scratch-wound assay

In scratch-wound assay, HGF induced narrowing of the wound while TGF-b1 showed less effect. Combined treatment with HGF and TGF-b1 resulted in enhanced cell migration compared with either HGF or TGF-b1 alone.

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Using the transwell chamber, co-treatment with HGF and TGF-b1 significantly induced cell migration compared with either HGF or TGF-b1 alone, confirming that the increase of cell migration probably was the cause ofenhanced wound healing by co-treatment with HGF and TGF-b1. While HGF induced wound healing better than TGF-b1 in scratch-wound, TGF-b1 induced far more transwell migration than HGF. It might be related withthe difference of the two assays: scratch-wound assay used attached cells, transwell assay used single cells

Hardwickeet al.[19]


Keratinocytes were incubated in DMEM (67.5%) andHams F12 (22.5%) with hydrocortisone (400 ng/ml),insulin (5 lg/ml) and 10% FBS. When cells reached 80–90% confluence, they were sub-cultured

rhEGF (6.25 ng/ml = 1 nM) Migration of cells and extent of woundclosure quantified by image analysis48 h

For scratch wound model, following trypsinization,keratinocytes were seeded into 24-well plates inmedium 1 ml with 10% FBS at 2.5 � 104 cells/well. Cellswere incubated for 48 h to reach 95–100% confluenceand then serum-starved for 24 h. Cells were washed 2�with PBS and medium was replaced with serum-freemedium containing EGF. A single linear scratch wasmade centrally across each cell monolayer using apipette tip. Cell migration was visualized over 48 h andextent of wound closure quantified by image analysis

Migration rate of keratinocytes measured over 48 h in absence and presence of EGF (6.25 lg/ml) was 7 and 28 lm/h, respectively

Ranzatoet al.[20]


Keratinocytes were grown to confluence, incubated for24 h in absence of serum, and then monolayers of cellswere scratched with a sterile 0.1–10 ll pipette tip. Afterwashing away suspended cells, cultures were refedwith medium in the presence or absence of HMGb1(high-mobility group box 1) protein. After 24 hincubation, cells were fixed in 3.7% formaldehyde in PBSfor 30 min, and then stained with 0.1% toluidine blue for30 min. Cell migration into the wound space wasestimated at 0 and 24 h after wounding by imageanalysis using an inverted microscope

To check for possible contribution of HMGb1 releasefrom wounded keratinocytes, the release of the proteinfrom dead cells was evaluated by overnight exposure to10 mM hydrogen peroxide. Also the levels of HMBGb1protein in the control and treated media were compared

rHMGb1 (10 nM) Migration of cells and extent of woundclosure estimated by image analysis24 h

Exposure to HMGb1 induced a significant increase of wound closure rate with respect to controls. The release of HMGb1 protein from wounded cells was much lower compared to the amount of HMGb1 addedexperimentally. The ERK1/2 inhibitor PD98059 (10 lM) abolished the effect of HMGb1 but did not affect the low closure rate of controls

Radek et al.[21]

HumankeratinocytesNHEK cell line

Primary keratinocytes were grown to confluency inserum-free Epilife medium. On day of experiment, cellswashed with PBS and pretreated with mitomycin C(10 lg/ml) 2 h before wounding to prevent cellularproliferation. After 2 h, mitomycin C was removed andcells washed 3� with PBS. Cells were then scratchedonce vertically with a pipette tip to create an artificialwound. Cells were washed twice to remove cellulardebris

Cells were stimulated by adding growth factor alone orwith DS (dematan sulfate). PBS was used as a negativecontrol (untreated cells). The same fields werephotographed every 8 h for a total of 24 h and areamigrated by keratinocytes calculated

rFGF-10 (10 ng/ml = 0.5 nM), rFGF-7(10 ng/ml = 0.5 nM) aloneand with DS (2 lM)

Migration of cells measured every 8 hfor a total of 24 h following woundingby a scratch

24 h

Keratinocytes stimulated with FGF-10 and FGF-7 alone did not exhibit a significant increase in migration compared to untreated cells. Keratinocytes stimulated with both FGF-10 and DS exhibited maximal migration ofgreater than 55% over a 24 h period compared with either FGF-10 or DS alone. There was a trend towards an increase in keratinocyte migration in the presence of FGF-7 and DS but was considerably less marked comparedwith FGF-10 and DS together

Peura et al.[22]


Keratinocytes were grown in DMEM with 5% FBS. Cellswere seeded at 5 � 104 cell/ml in 5% FBS medium andallowed to reach confluence for 4 days. The confluentmonolayers were serum starved after washing with PBS.All experiments were performed in the absence ofserum. After 1 day of serum starvation the monolayerswere scratch-wounded by the tip of a spatula, andwashed with PBS. Treatment with HGF was initiated1 day after wounding. Inhibitors were added to themonolayer cultures

rhHGF (25, 100 and 250 ng/ml = 0.5, 1.9 and 4.6 nM)3rd or 5th day after scratchwounding

The denuded area was measured on thethird and fifth day after wounding

Without HGF stimulation, keratinocyte migration achieved only approximately 5% wound healing, demonstrating a quiescent state of keratinocytes was achieved by prior serum starvation. The morphology of the cellsremained intact and cells remained adherent throughout the experiment. HGF dose-dependently induced wound healing by 7% at 25 ng/ml, 52% at 100 ng/ml and 62% at 250 ng/ml. The effect was maximum after 3 daysand did not change thereafter. HGF-induced scratch-wound healing was inhibited by phosphoinositide 3-kinase inhibitors (wortmannin and LY294002), and by inhibitors of the mitiogen-activated protein kinases MEK/ERK1/2 (PD98059 and U0126) and p38 (SB203580)

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Table 1 (continued)

Authors,reference



Brem et al.[23]

Humankeratinocytes

Primary keratinocytes were grown to 80% confluence inserum-free keratinocyte medium supplemented withEGF and bovine pituitary extract BPE (keratinocyte-SFM). At 24 h before experimentation, cells weretransferred to basal KBM (Gibco-BRL) and incubated ineither 0.09 or 1.2 mM CaCl2 to induce differentiation.Before being wounded, cell cultures were incubated inpresence or absence of 8 lg/ml mitomycin C for 1 h andwashed with PBS to inhibit proliferation. Scratchwounding of cell monolayers was performed usingsterile pipette tips. Cultures were incubated in presenceor absence of VEGF165 or EGF for 24 and 48 h

hVEGF165 (100 ng/ml = 2.6 nM)

The distance coverage by cells movinginto scratch-wound area wasquantified. Three images were analyzedper condition per time point

EGF (25 ng/ml = 4 nM)24 or 48 h

Keratinocytes grown in culture medium containing low Ca2+ (0.09 mM) resemble the activated keratinocyte phenotype, the cells that actively participate in reepithelization. Keratinocytes grown in high Ca2+ (1.2 mM)medium resemble differentiating keratinocytes. As their proliferation rate decreases, they form desmosomal junctions and start stratifying in culture, contributing minimally to reepithelization. VEGF165 and EGFstimulated migration of activated keratinocytes over the wound area. VEGF165 did not stimulate migration of differentiated keratinocytes, but EGF continued to promote their migration in scratch wound assay

Koivistoet al.[24]


Keratinocytes seeded on 24-well plates 2 � 105 cells/well in DMEM with 10% FBS

When growth factors were used as stimulants for cellmotility, cells were preincubated with inhibitors for 2 hprior to adding the growth factor to the cells. Confluentcell layers were scratch-wounded and treated withstaurosporine (30 nM), HB-EGF (1 ng/ml) or leftuntreated, alone or together with function-blockinganti-EGFR antibody (2 lg/ml), tyrphostin AG1478(300 nM) or function-blocking anti-HB-EGF antibody(10 lg/ml). The cells were allowed to migrate for 24 h

HB-EGF (1 ng/ml = 0.05 nM)

Migration of cells measured after 24 h

EGF (2 ng/ml = 0.32 nM)

Primary keratinocytes 1 � 105 cells/well seeded in KGM-2 medium. Cells were grown to confluence for 2 daysafter which the cultures were scratch-wounded with a200-ll pipette tip. Two perpendicular wounds werecreated. Loose cells removed by washing with PBS. Ca2+-free MEM with 1% Ca2+-free FBS in absence or presenceof staurosporine and inhibitors were added to the cells

After 24 h migration, the cells were fixed with 4%formaldehyde-5% sucrose and stained with crystal violet

TGF-a (3 ng/ml = 0.18 nM)

When cell rounding was examined, cells were fixed after2 h of HB-EGF stimulation

FGF-7 (2 ng/ml = 0.1 nM)TGF-b1 (10 ng/ml = 0.4 nM)24 h

Keratinocyte migration was induced by TGF-b1, FGF-7, HB-EGF, EGF, TGF-a and staurosporine and dependent on EGFR signaling, involving autocrine HB-EGF expression and potentially blocked by GSK-3 inhibitors SB-4152386 and LiCl. Inhibition of GSK-3 activity prevented cell rounding which is an early event in EGF-mediated keratinocyte migration

Satish et al.[25]

Humankeratinocytesneonatalforeskins

Keratinocytes 4 � 104/ml were plated in culture dishes.After reaching 80% confluence the cells were quiesced ingrowth factor deficient medium EpiLife medium for 48 hprior to being denuded by a rubber policeman at thecenter of the plate. The cells were treated with orwithout EGF (1 nM), IP-9 (IFN-c inducible protein 9,50 ng/ml), IP-10 (IFN-c inducible protein 10, 50 ng/ml),and CI-1 (calpain inhibitor 1, 5 lg/ml) and incubated for24 h. These concentrations provided either maximummotility or inhibition without toxicity. Photographswere taken at 0 and between 23 and 24 h, and therelative distance traveled by the cells at the acellularfront was determined by computer-assisted imageanalysis. The residual gap between migrating cells wasmeasured and expressed as a percentage of the area orwound remaining unfilled as normalized to EGF-inducedhealing of the wound

To confirm that cell proliferation was not confoundinganalyses, mitomycin C (0.5 lg/ml) was added at thetime of wounding: the results at 24 h wereindistinguishable in the presence or absence ofmitomycin C. The continuous inclusion mitomycin C(0.5 lg/ml) prevents cell proliferation in keratinocytes.For the undifferentiated keratinocytes the addition ofmitomycin C to the quiescent media had no appreciableeffect as there is no demonstrable increase in cellnumber for the 24 h period

EGF (1 nM) Migration of cells measured after 24 hIP-9 (50 ng/ml)IP-10 (50 ng/ml)24 h

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IP-9 (50 ng/ml) enhanced EGF-induced motility and calpain activity of undifferentiated keratinocytes, and also promoted motility (compared to no treatment) and calpain activity by itselfThe concentration used provided maximal motility stimulation, with half maximal being at approximately 25 ng/ml IP-9 promoting motility at approximately 20% of EGF-induced maximal level. Even at these lower

concentrations of IP-9 (10 and 25 ng/ml), the motility effect was additive to that of EGFIP-10 (50 ng/ml) acted similarly to promote keratinocyte motility and enhance the EGF-responseInhibition of calpain by CI-1 (5 lg/ml) prevented stimulation of motility of undifferentiated keratinocytes by either EGF or IP-9. CI-1 also abolished calpain activity triggered by IP-9

Haase et al.[12]


Primary keratinocytes were grown on ST3 fibroblastfeeder layer in FAD medium (DMEM:Ham’s F12 3:1).ST3 fibroblasts, strain J2, were cultured in DMEM with10% FBS

Keratinocytes were cultured on 60-mm cell culturedishes in the presence of 3T3 feeder cells or on type Icollagen-coated chamber slides without feeders. Whenthe cultures were confluent, remaining feeder cells wereremoved with PBS/EDTA and keratinocytes were kept incomplete FAD medium containing 50 lM Ca2+ (FAD lowCa2+) for 24 h. Cultures were treated with 4 lg/mlmitomycin C in DMEM containing 50 lg/ml Ca2+ (DMEMlow Ca2+) without FBS for 2 h, then washed in PBS andthe monolayer wounded with a tip of a glass pipette.Cells were incubated in DMEM low Ca2+ in the absenceor presence of growth factors and inhibitors for 4–6 h.Cells were fixed with 4% formaldehyde, permeabilizedwith 0.4% Triton X-100 for 5 min, and stained withTRITC-labelled phalloidin

EGF (10 and 100 ng/ml = 1.6 and 16 nM)

Digital microscopic images were takenof the denuded area

IGF-1 (100 ng/ml = 13 nM)4–6 h

Treatment with mitomycin C prevented formation of Ca2+-dependent cell–cell contacts and abrogated proliferation of keratinocytes. For the wounded keratinocyte monolayer, treatment with 10 or 100 ng/ml EGF acceleratedcell migration and wound closure compared with untreated control. Addition of 100 ng/ml IGF-1 also accelerated cell migration and wound closure. When EGF and IGF-1 were added simultaneously, repopulation of thedenuded area was faster than with either growth factor alone

In vitro assays of wound re-epithelialization showed that both growth factors stimulate migration of keratinocytes, and the activity of the respective signaling pathways was required for this re-epithelialization processEGF and IGF-1 influenced keratinocyte shape differently. IGF-1 stimulated membrane protrusion and facilitated cell spreading, whereas EGF induced contraction of keratinocytes. EGF treatment of keratinocytes stimulated

the mitogen-activated protein kinase (MAPK) pathway, and IGF-1 stimulated phosophatidylinositol-3-kinase (PI-3K). The results show that IGF-1 and EGF can influence different components of the keratinocyte migrationmachinery that determines the speed of wound epithelialization

Cha et al.[26]

Humankeratinocytesneonatalforeskin;tumorigenic SCC12F cells

Normal keratinocytes were cultured in keratinocyteserum-free growth medium (keratinocyte SFM) withEGF and bovine pituitary extract (BPE). SCC 12F cellswere maintained in 1:1 DMEM:Ham’s F12 mixture with5% iron-supplemented bovine serum

Cells were subcultured and maintained in growthmedium until colonies of greater than 16 cells wereestablished. Cells then deprived of EGF and BPE or serumfor 48 h before treatment with 10 nM EGF or TGF-a.Colony dispersion (cell scattering) was documented byphotography

rhEGF (10 nM) Colony dispersion measured after 48 h(normal keratinocytes); 24 h (SCC 12Fcells)

rhTGF-a (10 nM) mEGFColony dispersion: fornormal keratinocytes 48 h;SCC 12F cells 24 hMigration assay: fornormal keratinocytes 48 h

Migration of individual cells measuredfrom phagokinetic tracks produced bycells on colloidal gold-coated coverslips

For all experiments involving growth factor addition,keratinocytes transferred to keratinocyte SFM withoutEGF or BPE, and SCC 12F cells placed in DMEM:Ham’sF12 mixture containing 0.1% bovine serum albumin for48 h before growth factor addition

Confluent cell monolayers were deprived of serum andgrowth factors for 48 h, and a cell-free area wasintroduced by scraping the monolayer with a pipette tip,followed by washing to remove cellular debris.Repopulation of cleared area with cells over time wasmonitored. Parallel experiments were conducted in cellspretreated with mitomycin C (10 lg/ml) 2 h beforewounding to prevent cellular proliferation

TGF-a was more effective than EGF in promoting colony dispersion, in vitro wound closure by cell migration into denuded area, and single cell migration by phagokinetic track analysis

Tsuboiet al.[27]

Humankeratinocytesinfant foreskins

Keratinocytes were grown in keratinocyte growthmedium (KGM) containing EGF (10 ng/ml), insulin(5 lg/ml), CaCl2 (0.15 mM) and brain pituitary extract(0.4%v/v) in modified MCDB153 medium

For cell migration, cell suspensions of 2nd passagekeratinocytes in KGM (Ca2+ 0.08 mM) were seeded at6 � 104 cells/ml in 12-well plates to achieve confluencyquickly. After 48 h the medium was changed to KBM(Ca2+ 0.08 mM) and the cells were incubated for anadditional 6 h. Half of the plate’s confluent monolayersof keratinocytes were removed using a razor blade andcell scraper. The remaining keratinocytes wereincubated in KBM (Ca2+ 0.08 mM) for 16 h in presence ofgrowth factors. After incubation, the cells were fixedwith absolute methanol, stained with Giemsa, andcounted using a light microscope with occular grid

FGF-7 (1 nM) Number of cells migrating into 10successive 125 lm zones counted after16 h

FGF-2 (1 nM)TGF-a (1 nM)16 h

All experiments were carried out using 2nd or 3rdpassage keratinocytes grown in KGM. The effect ofadded growth factors was examined after the mediumwas replaced with serum-free keratinocyte basalmedium (KBM, Curabo Co, Osaka, Japan)

FGF-7 (1 nM) was as potent as TGF-a (1 nM) and more active than FGF-2 (1 nM) in stimulating keratinocyte migration


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Table 1 (continued)

Authors,reference



Tsuboiet al.[28]

Humankeratinocytesinfant foreskins

Keratinocytes were grown in keratinocyte growthmedium (KGM) containing EGF (10 ng/ml), insulin(5 lg/ml), CaCl2 (0.15 mM) and brain pituitary extract(0.4%v/v) in modified MCDB153 medium

For cell migration, keratinocytes were seeded in 12-wellplates and grown in KGM (Ca2+ 0.08 mM). The mediumwas switched from KGM to KBM 6 h prior to theexperiment. Half of the plate’s confluent monolayer ofkeratinocytes was removed with a razor blade, and theremaining keratinocytes were incubated in KBM for 24 hin the presence of indicated growth factors. Afterincubation, the cells were fixed with absolute methanol,stained with Giemsa, and counted using a lightmicroscope with occular grid

rTGF-a (1 nM) Number of cells migrating into 10successive 125 lm zones counted after24 h

All experiments were carried out using 3rd passagekeratinocytes grown in KGM (Ca2+ 0.08 mM) withoutcoating of plate. The effect of added growth factors wasexamined after the medium was replaced with serum-free keratinocyte basal medium (KBM, Curabo Co, Osaka,Japan)

rFGF-1 (1 nM)rFGF-2 (1 nM)

rFGF-7 (1 nM)rHGF (1 nM)rIGF-1 (1 nM)pTGF-b (1 nM)24 h

TGF-a, FGF-1, FGF-2, FGF-7, HGF and IGF-1 at 1 nM concentration stimulated migration of keratinocytes while TGF-b suppressed it. IGF-1 showed a modest effect compared with the other growth factors. HGF had the greateststimulatory activity among the tested growth factors, with the distance that the cells migrated from the wound edge as well as cell numbers being the greatest for HGF. The effects of FGF-7 and HGF on cell migration weredose-dependent

In Tables 1, 3 and 5, the concentrations of fetal bovine serum (FBS) and additives such as EGF, bovine pituitary extract (BPE) and insulin in culture medium have been indicated but not of antibiotics or fungicides.Where in the original article the concentration of growth factor or cytokine was reported in ng/ml, the concentration has been calculated in nM as this was considered to be more informative when comparing the influence ofdifferent growth factors or cytokines. The following estimated molecular weights were used: AGF, 70 kDa; EGF, 6.4 kDa; FGF-1, 17 kDa; FGF-2, 22 kDa; FGF-7, 19 kDa; FGF-10, 20 kDa; HB-EGF, 22 kDa; HGF, 54 kDa; HMGb1,25 kDa; IGF-1, 7.6 kDa; IL-8, 10 kDa; IP-9, 14 kDa; IP-10, 10 kDa; MGSA, 16 kDa; PDGF, 27 kDa; TGF-a, 17 kDa; TGF-b1, 25 kDa; VEGF165, 38 kDa.

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Table 2Levels of growth factors and cytokines tested for effect on keratinocyte migration in scratch-wound assay or similar procedure.

Growth factor or cytokine tested and concentration(ng/ml)

Reference Enhancedmigration

Comments

EGF0.1, 0.5, 1 Tochio et al. [16]

p,p

,p

6.25 Hardwicke et al. [19]p

25 Ca2+ 0.09, 1.2 mM Brem et al. [23]p

,p

2 Koivisto et al. [24]p

10, 100 Haase et al. [12]p

,p

64 Cha et al. [26]p

HB-EGF1 Koivisto et al. [24]

p

TGF-a TGF-a 170 ng/ml > EGF 64 ng/ml3 Koivisto et al. [24]

p

170 Cha et al. [26]p

6 Tsuboi et al. [27]p


TGF-b12 Rasanen and Vaheri

[17]

p

2 Nam et al. [18]p

10 Koivisto [24]p

25 Tsuboi et al. [28] x

HGF HGF 6 ng/ml + TGF-b1 2 ng/ml > HGF 6 ng/ml or TGF-b1 2 ng/ml

6 Nam et al. [18]p

25, 100, 250 Peura et al. [22] x,p

,p


FGF-710 Radek et al. [21] x10 + DS 2 lM Radek et al. [21] x2 Koivisto et al. [24]

p



FGF-1010 Radek et al. [21] x10 + DS 2 lM Radek et al. [21]

p

FGF-222 Tsuboi et al. [27]

p


FGF-117 Tsuboi et al. [28]

p

IGF-1 IGF-1 100 ng/ml + EGF 10 ng/ml > IGF-1 100 ng/ml or EGF10 ng/ml

100 Haase et al. [12]p


VEGF100 Ca2+ 0.09,1.2 mM Brem et al. [23]

p, x

HMGb1 Ranzato et al. [20]p

250IP-950 Satish et al. [25]

p

IP-1050 Satish et al. [25]

p


(19 ng/ml, 17 ng/ml and 22 ng/ml, respectively). In addition, FGF-1(acidic FGF) at 17 ng/ml stimulated keratinocyte migration.

Other growth factors examined were IGF-1 and VEGF. IGF-1 wastested in two studies and stimulated keratinocyte migration at 8and 100 ng/ml. When IGF-1 at 100 ng/ml was combined withEGF at 10 ng/ml, migration of keratinocytes into the wound areawas faster than with either growth factor alone. VEGF at 100 ng/ml increased the migration of keratinocytes in the presence oflow (0.09 mM) but not high Ca2+ (1.2 mM).

The high mobility group box 1 (HMGb1) protein enhancedkeratinocyte migration at 250 ng/ml, as did IFN-c inducible protein9 (IP-9) at 50 ng/ml and IP-10 at 50 ng/ml.

3.2. Migration of keratinocytes in Boyden and transwell chambers(Tables 3 and 4)

There were six studies of which five had used HaCaT cell lineand one used keratinocytes grown from donor tissue. Five of thestudies involved chemotaxis, and one examined chemokinesis.The extent of migration of cells from the upper chamber to thelower chamber was determined over a time period varying from6 to 24 h (mean 21 h).

The effect of TGF-b1 was determined in two of the studies. Thisgrowth factor at 2 ng/ml increased the migration of HaCaT cells inboth chemotaxic and chemokinetic assays. HGF at 6 ng/ml also

Table 3Migration of keratinocytes in Boyden and transwell chambers.

Authors,reference

Type of cells Culture medium and plating density of cells Cell culture Growth factor or cytokineadded to culture medium,and duration of treatment ofcells with growth factor orcytokine

Experimental outcomes and time when measuredafter growth factor or cytokine treatment

RasanenandVaheri[17]

HumankeratinocytesHaCaT cellline

Keratinocytes were cultured in DMEM with 5% FBS.For chemotaxis assay, cells were labeled with greenfluorescent protein (GFP). To test migration responseto TGF-b1, modified Boyden chambers with 3-lmpore polycarbonate filters were inserted in a 24-wellplate. The lower chamber was filled with controlmedium or TGF-b1-containing medium and GFP-labeled keratinocytes (5 � 104, 200 ll) added toupper chamber. Cells were let to migrate for 24 h,after which inserts were washed with PBS and non-migrated cells from top of the filter were removed.Migrated cells were photographed using an invertedimmunofluorescence microscope

To confirm that TGF-b1 causes proliferation arrest,keratinocytes were seeded in 96-well plates(2 � 104 cells/well) and let to plate for 2 h. Culturemedium with unattached cells was aspirated andreplaced with 100 ll of either control medium orTGF-b1. Cells were incubated for 48 h after whichMTT reagent (5 mg/ml) was added and incubated for4 h. Culture medium was aspirated and 100 ll ofcrystal dissolving solution was added to each welland absorbance measured at 540 nm. Levels ofepithelial junction proteins ZO-1 and E-cadherin incells in response to TGF-b1 were investigated byimmunoblotting and immunofluorescence

rhTGF-b1 (2 ng/ml = 0.08 nM) Migration ofcells: 24 h; Proliferationassay: 48 h; Levels ofepithelial junction proteins:48 h

Migration of cells measured at 24 h in Boydenchamber. Proliferation of cells measured by MTTassay at 48 h. Levels of epithelial junctionproteins at 48 h measured by immunoblottingand immunofluorescence

Comments on study design and findingsTGF-b1 caused proliferation arrest of keratinocytes. Expression of ZO-1 decreased in response to TGF-b1 treatment. E-cadherin level was downregulated to a lesser extentIn chemotaxis assay, TGF-b1 significantly increased the migration of keratinocytes

Namet al.[18]


Keratinocytes were grown in RPMI-1640 mediumwith 10% FBS

rhHGF (30units/ml � 6 ng/ml = 0.1 nM)

Migration of cells determined by counting cellson the lower surface of the filter of Transwell

In migration assay, cells were seeded (1 � 104 cells/well) into 24-well plates in RPMI-1640 with 0.1% FBSonto the microporous membrane (8.0 lm) in upperchamber of the transwell. After 24 h treatment withcytokines, the unmigrated cells in the upperchamber were gently removed using a cotton swab.Cells which had migrated through the membrane tothe lower chamber were fixed, stained with crystalviolet and washed. Migration was determined bycounting cells on the lower surface of the filter byphase-contrast microscopy

rhTGF-b1 (2 ng/ml = 0.08 nM)24 h migration assay

Using the transwell chamber, co-treatment with HGF and TGF-b1 significantly induced cell migration compared with either HGF or TGF-b1 alone, confirming that the increase of cell migration probably was the cause ofenhanced wound healing by co-treatment with HGF and TGF-b1. While HGF induced wound healing better than TGF-b1 in scratch wound, TGF-b1 induced far more transwell migration than HGF. It might be related withthe difference of the two assays: scratch wound assay used attached cells, transwell assay used single cells

Ranzatoet al.[20]


Cells 1 � 105 per well were added to the uppercompartment of transwell plates (8 lm pore size offilters). After 6 h filters removed and stained with0.5% crystal violet for 10 min and washed 3� withwater. The upper surface of filters was scraped usinga cotton swab to remove cells that had attached butnot migrated. Following PBS washing of filters, thedye was eluted from cells with 33% acetic acid andabsorbance measured at 540 nm

rHMGb1 (10 nM) Chemotaxis migration assay using transwellchambers6 h

HMGb1 at 10 nM for 6 h increased keratinocyte migration rate compared to controls. PD98059 blocked completely the stimulation of HMGb1 on keratinocyte migration, but it did not alter the migration rate of controls

Zhanget al.[29]


Cells were grown in DMEM:F12 mediumsupplemented with 10% FBS

rhAGF (2 and 4 lg/ml = 29and 57 nM)

Migration assays using transwell chambers

Cells were washed and adjusted to a density of4 � 105 cells/ml with DMEM:F12 or DMEM. Cells4 � 104 in 100 ll medium were added to the upperchamber of a 24-well transwell plate and allowed tomigrate for 20 h from upper chamber to lowerchamber and to which angiopoietin-related growth

20 h

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factor (AGF) had been added. Cells that had migratedthrough the membrane were then fixed in 4%paraformaldehyde for 30 min and stained by 0.5%crystal violet for 30 min. After extensively washing,the stained cells were solubilized with 500 ll of 1%SDS and OD 570 nm measured

AGF stimulated keratinocytes proliferation only at high protein concentration but promoted adhesion, spreading, and migration of keratinocytes. The nature of AGF-mediated migration was chemotactic, not chemokinetic

Sogabeet al.[30]


Keratinocytes were cultured in serum-free HuMedia-KG2 (keratinocyte growth medium, KGM). Cells wereused between passages 3 and 4. The same mediumwas used without BPE and hEGF for maintenance ofkeratinocytes (keratinocyte basal medium KBM)

Cell migration assays were performed using Boydenchamber with transwell inserts. Subconfluentkeratinocytes were detached from the dishes bytrypsinization, resuspended in KGM, and diluted to afinal cell concentration at 1.5 � 105 cells/ml. Thebottom wells of a 12-well Boyden chamber werefilled with KGM. To allow cells to adhere on theupper surface of the filter membrane (12 lm poresize polycarbonate membrane), 500 ll ofkeratinocyte suspension was added to each upperwell of the Boyden chamber, and the chamberincubated for 24 h. To start the migration assay, themedium in bottom wells was removed and changedto KBM containing one of the following: 10% FBS,bFGF (1 ng/ml), KGF (0.1, 1, and 10 ng/ml). Thechambers were further incubated for 12, 18 or 24 h.Cells attached to the membrane were fixed with 2.5%glutaraldehyde for 15 min, and stained with Mayer’shematoxylin. The membranes were cut off from theupper chamber and mounted on glass slides withFluoromount G. The number of keratinocytesattached on upper or lower side of the membranewas quantified in 3 randomly chosen fields permembrane.

Cell migration: hrFGF-2(1 ng/ml = 0.05 nM)

Keratinocyte migration in Boyden chambers.

hrFGF-7 (0.1, 1, 10 ng/ml = 0.005, 0.05, 0.5 nM)Cell migration assay: 12, 18and 24 h

Keratinocyte migration was significantly increased in the presence of 1 ng/ml FGF-2 or 10 ng/ml FGF-7 at 12, 18 and 24 h

Kurschatet al.[31]

HumankeratinocytesHaCaT celllines

Keratinocytes were maintained as monolayers inMEM containing 10% FBS. Cells were cultured for24 h. For migration assay, 1 � 104 cells in serum-freemedium were seeded into upper chamber oftranswell inserts (the filter had a pore size 12 lm).Fully supplemented MEM medium with 10% FBS wasadded to the lower compartment as thechemoattractant. VEGF165 was added to the media ofboth compartments at concentration of 10 ng/ml.After 16 h cells on lower side of filter were stainedwith xanthine/thiazine dyes and counted

VEGF165 (10 ng/ml = 0.26 nM) 16 h

On each filter five fields of a defined size evenlydistributed over the filter were counted

VEGF165 did not affect the proliferation or the migration of any of the keratinocyte HaCaT cell lines examined. These were wild type cells, cells overexpressing neuropilin-1 (NRP1), and cells overexpressing neuron restrictivesilencer factor (NRSF)

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Table 4Levels of growth factors and cytokines tested for effect on keratinocyte migration in Boyden and transwell chambers.

Growth factor or cytokine tested and concentration (ng/ml)

Reference Enhancedmigration

Comments

TGF-b12 Rasanen and Vaheri

[17]

pChemotactic

2 Nam et al. [18]p

Chemokinetic TGF-b1 2 ng/ml > HGF 6 ng/ml

HGF HGF 6 ng/ml + TGF-b1 2 ng/ml > HGF 6 ng/ml or TGF-b12 ng/ml6 Nam et al. [18]

pChemokinetic

FGF-70.1, 1, 10 Sogabe et al. [30] x, x,p

Chemotactic

FGF-21 Sogabe et al. [30]

pChemotactic

AGF2, 4 Zhang et al. [29]

p,p

Chemotactic

VEGF10 (both chambers) FBS chemoattractant (lower

chamber)Kurschat et al. [31] x

HMGb1250 Ranzato et al. [20]

pChemotactic


enhanced chemokinetic migration of HaCaT cells but was less thanthat induced by TGF-b1 at 2 ng/ml. Combined treatment with HGFwith TGF-b1 resulted in significantly increased migration of cellscompared to either growth factor alone.

Two members of the fibroblast growth factor family, FGF-2 andFGF-7, were tested in chemotactic assay. FGF-2 at 1 ng/ml and FGF-7 at 10 ng/ml increased keratinocyte migration, while FGF-7 at 0.1or 1 ng/ml was without significant effect.

Angiopoietin-related growth factor (AGF) at 2 and 4 lg/ml in-creased the chemotactic migration of HaCaT cells. By contrast, nosignificant increase in migration occurred when VEGF at 10 ng/ml was added to both compartments and with 10% fetal bovineserum (FBS) as a chemoattractant in the lower compartment.

HMGb1 at 250 ng/ml increased the chemotactic migration rateof HaCaT cells.

3.3. Cell shape, cell spreading, and migration of keratinocytes on ECMproteins (Tables 5 and 6)

Seven studies were found and all had used keratinocytes fromdonated tissues. In all of the studies the cells were starved priorto the experiment. One study reported on cell shape and spreading,while the others had investigated cell migration.

Keratinocytes spread bidirectionally with a prominent develop-ment of actin stress fibers and formed lamellipodia when stimu-lated with FGF-2 at 1 ng/ml on type I collagen-coated coverslipsfor 4 h. Cells treated with FGF-7 at 10 ng/ml extended in all direc-tions on type I collagen-coated coverslips. On fibronectin-coatedcoverslips, the shape of keratinocytes treated with FGF-2 at 1 ng/ml or FGF-7 at 10 ng/ml was polygonal.

In the studies investigating the effects of growth factors onkeratinocyte motility on culture dishes coated with ECM proteins,the migration of cells was followed over a time period rangingfrom 16 to 24 h (mean 20 h). Keratinocyte migration was acceler-ated on fibronectin, laminin and vitronectin, but was similar tocontrols on type I collagen and type IV collagen.

There was no significant increase in migration of keratinocyteswith interleukin-8 (IL-8) at 1, 10 or 100 ng/ml on dishes coatedwith type IV collagen, laminin or fibronectin, and migration was re-duced with IL-8 at 100 ng/ml on these proteins.

A significant increase in keratinocyte migration occurred withmelanoma growth stimulatory activity (MGSA) at 1 ng/ml on lam-inin-1, but MGSA at higher doses of 10 and 100 ng/ml brought

about significantly reduced migration. Migration on type IV colla-gen was not changed by MGSA at any of the doses used, whileMGSA at 10 and 100 ng/ml caused significantly lowered migrationon fibronectin.

EGF and TGF-a at 0.001–1 ng/ml increased migration of kerati-nocytes on type I collagen, type IV collagen and fibronectin,whereas very low levels of migration occurred on laminin evenwith high doses of EGF. EGF at 60 ng/ml and TGF-a at 170 ng/mlincreased migration on fibronectin. FGF-2 at 1 ng/ml stimulatedmigration on type I collagen. PDGF at 10 ng/ml stimulated migra-tion of keratinocytes on serum-treated dishes. None of the growthfactors TGF-b, nerve growth factor (NGF), IL-6, and IL-8 influencedcell migration on type I collagen, type IV collagen, fibronectin orlaminin.

IL-1a at 0.1–10 U/ml produced a significant increase in migra-tion on type I collagen and type IV collagen but not on laminin.The addition of both IL-1a and TGF-a at concentrations suboptimalfor migration (such as IL-1a at 0.1 U/ml and TGF-a at 0.01 ng/ml)produced significantly greater migration on type I collagen as com-pared to the addition of either cytokine alone. However when eachcytokine was added at optimal concentration, no additional in-crease in migration was found beyond that seen for optimal con-centration of the first cytokine with further addition of thesecond cytokine.

Both EGF at 1–50 ng/ml and IGF at 1–50 ng/ml enhanced kerat-inocyte migration on wells coated with colloidal gold and type IVcollagen. The effect of EGF on migration was concentration depen-dent with a maximum at 10–50 ng/ml EGF.

4. Discussion

4.1. Research design and reporting of studies

The largest number of studies examining keratinocyte migra-tion was performed using a scratch wound assay or similar proce-dure and on coverslips and dishes with an ECM protein coating.Only a few studies have been performed using Boyden or transwellchambers. A total of 21 growth factors and cytokines have beentested for effect by these methods with some being tested in morethan one method, enabling a comparison of effect in the differentassays. The cells used were keratinocytes derived from donatedhuman tissue (e.g. neonatal foreskin) or the cell lines HaCaT andNHEK. All of the studies used cells cultured in serum-free or low

Table 5Cell shape, cell spreading and migration of keratinocytes on coverslips and dishes coated with ECM protein.

Authors,reference


Experimental outcomes and timewhen measured after growth factor orcytokine treatment

Sogabe et al.[30]

Human keratinocytesneonatal foreskins

Keratinocytes were cultured in serum-freeHuMedia-KG2 (keratinocyte growth medium,KGM). Cells were used between passages 3 and 4.The same medium was used without BPE andhEGF for maintenance of keratinocytes(keratinocyte basal medium KBM)

Cell spreading: hrFGF-2(1 � 10�3–1 � 103 ng/ml = 0.000045–45 nM)

Shape and spreading of keratinocytesby immunofluorescence microscopyof cells stained for actin stress fibersand vinculinhrFGF-7 (0.1–10 ng/

ml = 0.005–0.5 nM)Cell migration: hrFGF-2(1 ng/ml = 0.045 nM)

Subconfluent keratinocytes were detached fromthe dishes by trypsinization, and resuspended inKBM

hrFGF-7 (0.1, 1, 10 ng/ml = 0.005, 0.05, 0.5 nM)

To observe the effects of bFGF or KGF onkeratinocyte spreading, cells were seeded at4 � 105 cells on 18 mm square glass coverslipswhich were coated both with and without type Icollagen or fibronectin. For the purpose of coating,coverslips were placed in 6-well dishes and eithertype I collagen 50 lg/ml or fibronectin 2 lg./mlwas added before incubation for 30 min, and rinsein KBM. To allow the cells to attach to thecoverslips, cells were incubated for 15 min andthen incubated with one of the following in KBMfor 4 h: 10% FBS, bFGF and KGF. Cells were fixedwith 3% paraformaldehyde and then processedand stained to visualize actin and vinculin byimmunofluorescence microscopy

Cell spreading: 4 h

Comments on study design and findingsKeratinocytes spread bidirectionally and formed lamellipodia only when they were stimulated with 1 ng/ml FGF-2 on type I collagen-coated coverslips. Keratinocytes treated with 10% FBS or 10 ng/ml FGF-7 extended in all

directions. On fibronectin-coated coverslips, keratinocytes treated with 10% FBS extended in all directions, while with 1 ng/ml FGF-2 or 10 ng/ml FGF-7 the shape of keratinocytes was polygonal. FGF-2 exerts a stimulatoryeffect on keratinocyte migration in the presence of type I collagen as a scaffold

Sutherlandet al. [32]

Human keratinocytes tissuesamples fromauriculoplasty,abdominoplasty andmammoplasty

Epidermis was digested using 0.25% trypsin–EDTAsolution for 10 min, centrifuging 5 min. Cells weremaintained using serum-free MCDB 153 mediumcontaining 10 ng/ml EGF, 5 lg/ml insulin, 2.5 lg/ml BPE

For analysis of cell-substrate adhesion and celldivision, culture dishes were derivatized usingfibronectin, type I collagen, type IV collagen,laminin, vitronectin, and ECM gel for 2 h at roomtemperature before rinsing 3� in water.Keratinocytes at passage 1, 2 and 3 wereinoculated into dishes at 1 � 104 cells/ml and after6 days numbers of cells present in 6 fields of viewselected by systematic random sampling werecounted

PDGF (10 ng/ml = 0.4 nM) Time lapse clips converted intomovies and still image series andmovement of cells plotted

FGF-1 (10 ng/ml = 0.6 nM)EGF (10 ng/ml = 1.6 nM)TGF-b (10 ng/ml = 0.4 nM)FGF-7 (10 ng/ml = 0.5 nM)Cell-substrate adhesionand proliferation: up to6 daysCell movement: 24–72 h

For investigation of growth factor effects, PDGF,FGF-1, EGF, TGF-b, KGF were included in themedia. Keratinocytes were inoculated into serum-treated dishes at 1 � 104 cells/ml. Cultures werefilmed for 24–72 h

None of the ECM-derivatized substrata increased adhesion of keratinocytes over an 8 h periodKeratinocytes lost their proliferative potential following the first passage. Proliferation of keratinocytes was accelerated on fibronectin which induced a 10� increase in cell numbers by day 6, and on vitronectin; type I

collagen, type IV collagen, laminin, and ECM gel had no significant effect. Keratinocyte proliferation was accelerated by PDGF, EGF and FGF-7TGF-b and FGF-1 did not increase keratinocyte proliferation compared with controlsKeratinocytes showed increased motility with passage, the increase in motility being reflected by the alteration in morphology of cells, with the smallest and most rounded cells being the most active. Keratinocyte migration

was accelerated on fibronectin, laminin and vitronectin, but was similar to controls on type I collagen and type IV collagen


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Table 5 (continued)

Authors,reference



Chemokinesis of keratinocytes was induced by PDGF, EGF, FGF-7, and TGF-b but not by FGF-1. Of these, EGF was most effective with cells having a mean velocity almost twice that of control. The rounded keratinocytes werefar more motile than any other cell type

Draper et al.[33]

Human keratinocytes NHEKcell line and A431 cells (anepidermoid carcinoma cellline expressing very highlevels of EGFR)

NHEK cells were cultured in KGM with 0.1 lg/mlEGF, 5 mg/ml insulin, 0.5 mg/ml hydrocortisone,7.5 mg/ml BPE

NHEK or A431 cells were subcultured andmaintained in their respective growth media insingle well chamber slides coated with 10 lg/mlfibronectin. Following 24 h of serum deprivation,confluent monolayers were scratched with apipette tip in a standardized manner to createuniform cell free wounds in the monolayers. Themonolayers were washed vigorously 3� withDulbecco’s PBS to remove cell debris. Woundedmonolayers were then incubated for either 24 h inserum free medium alone as control, or with theaddition of EGF or TGF-a. Monolayers werewashed 2� in DPBS, fixed for 5 min in 4%paraformaldehyde and stained with hematoxylinand eosin. Wounded monolayers werephotographed and the extent of wound closuremeasured. To inhibit cell proliferation,experiments were conducted with the addition of10 lg/ml mitomycin-C throughout the duration ofthe experiment

hrEGF (0–250 ng/ml = 0–40 nM)

Wound closure measured at 24 or72 h

hrTGF-a (0–700 ng/ml = 0–41 nM)24 or 72 hA431 cells cultured in DMEM:Ham’s F12 mixture

(1:1) with 10% FBSIn all experiments involving addition of growthfactors, NHEK were washed free of KGM andplaced in growth factor and serum free KBM for 24or 72 h before growth factor addition. A431 cellswere washed free of serum containing mediumand placed in serum free DMEM:F12

For wounded NHEK monolayers, treatment with 60 ng/ml EGF for 72 h caused 69% wound closure. For wounded A431 monolayers, treatment with EGF for 24 h induced progressive dose related increases in wound closure atconcentrations 0–60 ng/ml, with maximal response at 60 ng/ml. EGF dose dependent inhibition of wound closure occurred at concentrations of 120 ng/ml and higher. TGF-a induced progressive dose related increases inwound closure at concentrations 0–170 ng/ml, with maximal response at 170 ng/ml. TGF-a dose dependent inhibition of wound closure occurred at concentrations of 340 ng/ml and higher. Addition of mitomycin-C towounded A431 monolayers treated with 60 ng/ml EGF or 170 ng/ml TGF-a for 24 h resulted in a non-significant 9% and 25% decrease in total wound closure respectively, indicating that cell proliferation plays a minor rolein the promotion of wound closure by these two growth factors

Rennekampffet al. [34]

Human keratinocytescadaveric donor skin

Cells were grown in serum-free medium(keratinocyte SFM) and released from cultureflasks by trypsinization

rhIL-8 (1, 10 and 100 ng/ml = 0.1, 1 and 10 nM)

Migration measured by counting thenumber of cells that migrated intoseparate fields (each field 1 � 1 mm).At least six different fields wereevaluated

24 h The number of migrated cellsstimulated with IL-8 was divided bythe number of migrated control cellsfrom the same donor. Migration indexof control cells set as 100%

Culture dishes (60 � 15 mm) were coatedovernight with either type IV collagen (9 lg/ml),laminin-1 (2 lg/ml) or fibronectin (3 lg/ml). Asmaller culture dish (40 � 10 mm) was placedinverted into the larger culture dish. Keratinocyteswere grown in the larger coated culture dishsparing the area of the inverted culture dish.Keratinocytes were grown to confluence usingkeratinocyte SFM medium. After confluence, theinverted smaller culture dish was removed and themedium was changed to keratinocyte SFM(control) or keratinocyte SFM with IL-8. After 24 hthe cells which had migrated into the spared areawere counted using a phase-contrast microscope

No significantly increased keratinocyte migration indices were found for any matrix or dosage tested of IL-8 compared with unstimulated keratinocytes. A decrease in migration of keratinocytes on laminin-1 occurred with100 ng/ml IL-8 (10 ± 9%, mean ± SE, of value for control unstimulated keratinocytes). Migration of keratinocytes on fibronectin was significantly reduced at 100 ng/ml IL-8 (15 ± 7%). For collagen type IV, migration ofkeratinocytes was lowered at 100 ng/ml IL-8 (65 ± 15%)

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Rennekampffet al. [35]

Human keratinocytescadaveric donor skin

Cells were grown in serum-free medium(keratinocyte SFM) and released from cultureflasks by trypsinization

rhMGSA (1, 10 and100 ng/ml = 0.06, 0.6 and6 nM)

Migration measured by counting thenumber of cells that migrated intoseparate fields (each field 1 � 1 mm).At least 6 different fields wereevaluated

24 h The number of migrated cellsstimulated with IL-8 was divided bythe number of migrated control cellsfrom the same donor. Migration indexof control cells set as 100%

Culture dishes (60 � 15 mm) were coatedovernight with either type IV collagen (9 lg/ml),laminin-1 (2 lg/ml) or fibronectin (3 lg/ml). Asmaller culture dish (40 � 10 mm) was placedinverted into the larger culture dish. Keratinocyteswere grown in the larger coated culture dishsparing the area of the inverted culture dish.Keratinocytes were grown to confluence usingkeratinocyte SFM medium. After confluence, theinverted smaller culture dish was removed and themedium was changed to keratinocyte SFM(control) or keratinocyte SFM with MGSA. After24 h the cells which had migrated into the sparedarea were counted using a phase-contrastmicroscope

Nonstimulated keratinocytes showed greater migration on type IV collagen than on laminin-1. Only on laminin-1 was a significant increase (1.9�) observed with 1 ng/ml MGSA stimulation. On laminin-1 higher doses of10 ng/ml and 100 ng/ml MGSA showed significantly reduced migration indices. Migration on type IV collagen was not significantly changed by MGSA at any of the dosages given. On fibronectin, 10 ng/ml and 100 ng/mlMGSA-stimulated keratinocytes showed significantly reduced migration indices

Chen et al.[36]


Keratinocytes were subcultured and passaged inserum-free keratinocyte growth medium (KGM).Cells were subsequently passaged twice to removeany contaminating fibroblasts before use.Keratinocyte migration on selected ECMcomponents was quantitated by computer-assisted image analysis. Preliminary experimentsdemonstrated the following optimal platingconcentrations of matrix components for maximaleffect on keratinocyte migration: type I collagen,15 lg/ml; type IV collagen, 15 lg/ml; fibronectin,60 lg/ml; laminin, 90 lg/ml. For selectedexperiments, suboptimal amounts of matrix andgrowth factors were used to determineconcentration-dependent effects of IL-1a. Afteradsorption of matrix molecules, coverslips werewashed and a suspension of keratinocytes wasadded to each well along with any soluble factorsto be tested. The culture plates were incubated forup to 24 h. The cells were fixed with 0.1%formaldehyde at selected times during the assay

The doubling time of keratinocytes under theseconditions was approximately 30 h, so that theassay directly assessed only cellular motilityunconfounded by the possibility of cell division

rIL-1a (0.1–10 U/ml) Images of 10 non-overlapping fieldsfrom each duplicate set of wellscaptured

rTGF-a (0.1 ng/ml = 0.006 nM) up to 24 h

The addition of rIL-1a to keratinocytes produced a statistically significant and concentration-dependent increase in migration on matrices of type I collagen and type IV collagen, but not on laminin. Maximal levels ofkeratinocyte migration obtained on these matrices with IL-1a were comparable to those obtained with stimulation by EGF and TGF-a. The effects of TGF-a and IL-1a on keratinocyte migration are additive; however, themaximal level of migration achieved by using IL-1a and TGF-a in combination never exceeds the maximal level of migration found by using either cytokine alone. No significant difference in migration when cells allowedto migrate for 16 h on type I collagen (4 lg/ml) or type IV collagen (4 lg/ml) with or without TGF-a at 0.001 ng/ml or 0.1 ng/ml in presence of IL-1a at 0.1 U/ml

Time course of keratinocyte migration induced by IL-1a was delayed (onset of migration 9–12 h after addition) as compared with that induced by TGF-a (onset of migration 6–9 h after addition) even if the cells werepreincubated in IL-1a

Chen et al.[37]


Keratinocytes were subcultured and passaged inlow Ca2+ and serum-free keratinocyte growthmedium (KGM). Cells were passaged twice toremove any contaminating fibroblasts.Keratinocyte migration was quantitated bycomputer-assisted image analysis. Coverslips withimmobilized gold salts were placed in 35-mm

The doubling time of keratinocytes under theseconditions was approximately 30 h, so that theassay directly assessed only cellular motilityunconfounded by the possibility of cell division

rhEGF (0.001–1 ng/ml = 0.00016–0.16 nM)

Images of 10 non-overlapping fieldsfrom each duplicate set of wellscapturedrTGF-a (0.001–1 ng/

ml = 0.00006–0.06 nM)TGF-bFGF-2NGF


P.V.Peplow

,M.P.Chatterjee

/Cytokine62

(2013)1–

2115

Table 5 (continued)

Authors,reference



culture dishes. The coverslips were then washedand 2 ml of HBSS supplemented with Ca2+ andMg2+ was added to each dish, along with any ECMmolecules to be tested. The following optimalplating concentrations of these matrixcomponents yielded maximum effects onkeratinocyte migration: type I collagen, 15 lg/ml;type IV collagen, 15 lg/ml; fibronectin, 60 lg/ml;laminin, 90 lg/ml. After adsorption of matrixmolecules, coverslips were washed and asuspension of keratinocytes added to each wellalong with any soluble factors to be tested. Cultureplates were incubated for 16 h. Cells were fixed,observe, photographed under dark field optics

IL-6IL-8 at concentrationsknown to elicit biologicalresponses in other types ofcells

Keratinocytes were cultured in keratinocyte basalmedium with 0.08 mM Ca2+. Migration ofkeratinocytes on matrices of type 1 collagen (4 and15 lg/ml) and fibronectin (60 lg/ml) in partiallysupplemented KBM

16 h

Keratinocytes cultured in KBM supplemented with 5 lg/ml insulin and apposed to an optimal matrix of type I collagen (15 lg/ml) exhibited very low levels of migration. Both rEGF and TGF-a at 0.001–1 ng/ml increasedmigration of keratinocytes apposed to connective tissue matrices of type I or type IV collagen or fibronectin. Migration occurred in a concentration-dependent manner. Keratinocytes apposed to connective tissue matricesof laminin or in the absence of connective tissue matrices exhibited very low levels of migration even in the presence of high concentrations of rEGF. None of the other growth factors influenced keratinocyte migrationeither in the presence or absence of matrix molecules. Blocking the EGF/TGF-a receptor on the cell surface of keratinocytes with specific antibody inhibited the stimulation of keratinocyte locomotion by rEGF and TGF-a.The growth-independent stimulation of keratinocyte locomotion via regulation of integrin expression may be one mechanism by which EGF accelerates the reepithelialization of human cutaneous wounds

Ando andJensen[38]


Cells were grown in serum-free MCDB 153 basemedium with 30 lM Ca2+ and BPE (140 lg/ml),insulin (5 lg/ml), mouse EGF (10 ng/ml), highamino acids. This medium is referred to ascomplete MCDB medium. Cells were harvestedfrom 50% to 80% confluent keratinocyte culturesbetween passages 2 and 4. Cells were thensuspended in MCDB(�) medium with 20 lg/mlBSA at 9 � 103 cells/ml; 0.4 ml of cell suspensionwas added to each well, which already contained0.4 ml of medium and when indicated appropriategrowth factors to be tested. Cells were incubatedon colloidal gold-coated plates for 2–23 h. At endof incubation, cells were fixed with 3%formaldehyde and observed under dark-fieldillumination. Experiments were repeated usingcolloidal gold plates coated with type IV collagen

EGF (0.1–50 ng/ml = 0.016–7.8 nM)

Three fields were photographed andareas of displaced gold was outlinedaround 30 cells. Total area ofmigration by these cells wasmeasured using an image analyzer

IGF-1 (1–50 ng/ml = 0.13–6.6 nM) 2–23 h

MCDB(�) medium refers to MCDB 153 basemedium with high amino acids but withoutgrowth factors

Addition of EGF to defined medium in the absence of any other growth factor induced an increase in migration of 2.5–4.5-fold on wells coated with colloidal gold and type IV collagen after overnight incubation; the effect ofEGF on migration was concentration dependent, with a maximum at 10–50 ng/ml EGF. Concentration-dependent enhancement of keratinocyte migration was similarly observed with IGF-I as well as with insulin on wellscoated with colloidal gold and type IV collagen after overnight incubation

With all factors, migration was observed on colloidal gold plates coated with type IV collagen or fibronectin but not in the absence of matrix coating. IGF-I and insulin enhance keratinocyte migration by a mechanism distinctfrom that of EGF

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Table 6Levels of growth factors and cytokines tested for effect on keratinocyte migration on ECM protein substrate.

Growth factor or cytokine tested andconcentration (ng/ml)

Reference ECMprotein

Enhancedmigration

Comments

EGF60 Draper et al.

[33]Fibronectin

p

0.001–1 Chen et al. [37] Type Icollagen

p

Type IVcollagen

p

Fibronectinp

Laminin Very low0.1–50 Ando and

Jensen [38]Type IVcollagen

p

TGF-a170 Draper et al.

[33]Fibronectin

p

0.001–1 Chen et al. [37] Type Icollagen

p

Type IVcollagen

p

Fibronectinp

Laminin Not indicated

TGF-bConcentration not reported Chen et al. [37] Type I

collagenx

Type IVcollagen

x

Fibronectin xLaminin x

FGF-21 Sogabe et al.

[30]Type Icollagen

p

Concentration not reported Chen et al. [37] Type Icollagen

x

Type IVcollagen

x


IL-8 Migration was reduced with IL-8 at 100 ng/ml on these matrices1, 10, 100 Rennekampff

et al. [34]Type IVcollagen

x, x, x

Fibronectin x, x, xLaminin x, x, x

Concentration not reported Chen et al. [37] Type Icollagen

x

Type IVcollagen

x


IL-6Concentration not reported Chen et al. [37] Type I

collagenx

Type IVcollagen

x


IL-1a No difference in migration on type I or type IV collagen with IL-1a 1U/ml ± TGF-a 0.001 or 0.1 ng/ml1 U/ml Chen et al. [36] Type I

collagen

p

Type IVcollagen

p

Fibronectin not indicatedLaminin x

IGF1–50 Ando and

Jensen [38]Type IVcollagen

p

NGFConcentration not reported Chen et al. [37] Type I

collagenx

Type IVcollagen

x

Fibronectin x



Table 6 (continued)

Growth factor or cytokine tested andconcentration (ng/ml)

Reference ECMprotein

Enhancedmigration

Comments

Laminin x

MGSA1, 10, 100 Rennekampff

et al. [35]Type IVcollagen

x, x, x

Fibronectinp

, x, xLaminin-1

p, x, x


FBS-containing medium prior to experimentation, and in the stud-ies using scratch wound assay or similar procedure the cells weregrown to complete or almost complete confluence. Thus the rate ofcellular proliferation would have been extremely low. In addition,some studies used prior treatment with mitomycin C to arrest cel-lular proliferation, so that the findings for example on the extent ofwound closure in scratch assay would not be confounded by cellu-lar proliferation. The effect of mitomycin C on wound closure inthese studies was tested and the results at 24 h were indistinguish-able in the presence or absence of mitomycin C at 0.5 lg/ml [25].Hydroxyurea is also known to have an anti-proliferative effectand has been used in migration studies of different types of cells(e.g. fibroblasts, endothelial cells). Hydroxyurea at 2 mM com-pletely inhibited the proliferation of human umbilical vein endo-thelial cells and had limited effect on migration [39], while at5 mM it inhibited both proliferation and migration [40].

4.2. Experimental procedures

The scratch wound assay and the procedure using coverslipsand dishes coated with ECM proteins are relatively easy to performand provide a useful screening of substances to be tested for effecton migration of cells. The cells are attached to the dishes or to theprotein coating of the coverslips or dishes. The method using Boy-den or transwell chambers is also easily performed and enables ameasurement of chemokinetic or chemotactic migration depend-ing on whether the activator is incorporated into the medium ofthe upper chamber to which the cells have been added or is withinthe medium of the lower chamber. A porous filter (3, 8 or 12 lmpore size) separates the two chambers and the cells areunattached.

Other methods have been reported as being superior to thescratch wound assay. Although the scratch assay is adequate forqualitative assessment of activators or inhibitors, it does not pro-vide the highly reproducible results necessary for quantitativecompound structure–activity relationship evaluation owing tothe inconsistent size and placement of the wound area withinthe microplate well. An alternative to the scratch assay is the Oriscell migration assay and enables the formation of accurately placedand consistently sized cell-free areas into which migration can oc-cur without releasing factors from wounded or dead cells or dam-aging an underlying protein coating [41].

4.3. Types of cells used

Most of the studies using the scratch assay or the transmigra-tion chamber assay had used the HaCaT cell line. This differs fromthe studies performed on ECM protein coatings of coverslips ordishes that had all used keratinocytes derived from donor tissues.While it is not known whether there are any differences in migra-tion in response to certain growth factors or cytokines for the Ha-CaT cell line compared to keratinocytes derived from donatedtissue (e.g. infant foreskins or tissue removed at surgery), it wouldclearly be advantageous for all the studies to be performed with

the latter cells. In this way the findings would have greater rele-vance to the reepithelization of wounds in vivo and to designingfuture strategies to promote the healing of chronic wounds. Alsoit would allow a much more meaningful comparison of findingson migration using the various assay methods. The NHEK cell linewas used only in one study for the scratch assay and one study ofmigration on slides coated with fibronectin.

4.4. Influence of growth factors and cytokines on keratinocytemigration

Members of various families of growth factors and cytokineswere tested for effect on cell migration in one or several of the as-says. They included EGF, TGF-a, HB-EGF belonging to the EGF fam-ily; FGF-1, FGF-2, FGF-7, FGF-10 of the FGF family; VEGF of VEGFfamily; PDGF of PDGF family; IGF-1 of IGF family; AGF of angio-poietin-related protein (ANGPTL) family; IL-1a, IL-6, IL-8 of IL fam-ily; MGSA of a-chemokine family; as well as TGF-b1, HGF, NGF,HMGb1, IP-9 and IP-10. It was noted that placental growth factorwhich belongs to the VEGF family had not been tested in any ofthese assays.

In the scratch wound assay, all the growth factors tested areknown to stimulate wound repair in vivo. Many were tested at dif-ferent concentrations, with some being very low and shown to en-hance cell migration. For example, EGF was effective at 0.1 and0.5 ng/ml (0.016 and 0.084 nM). It is noteworthy that EGF at25 ng/ml was effective in stimulating migration in the presenceof both low and high Ca2+ concentration (0.09 and 1.2 mM, respec-tively) while VEGF at 100 ng/ml stimulated migration in the pres-ence of low but not high Ca2+ concentration. TGF-a at 170 ng/ml(10 nM) was more effective than EGF at 64 ng/ml (10 nM) inenhancing migration.

It is known that two growth factors in combination may bemore stimulatory for wound repair in vivo than either growth fac-tor applied to the wound alone. In the scratch assay, HGF at 6 ng/ml in combination with TGF-b1 at 2 ng/ml was more effective instimulating migration than either HGF or TGF-b1 alone. Also IGF-1 at 100 ng/ml together with EGF at 10 ng/ml caused a greater in-crease in migration than IGF-1 or EGF alone. It would be helpful toknow whether other growth factors are more stimulatory whentested in pairs rather than singly e.g. FGF-2 with TGF-b, PDGF withTGF-a, PDGF with IGF-2, and FGF-2 with IGF-2 [42]. Furthermore,some growth factors had a more pronounced effect on migrationwhen tested in the presence of a soluble ECM proteoglycan. Forexample, FGF-10 at 10 ng/ml had little if any stimulatory effectby itself but in the presence of dermatan sulfate at 2 lM it en-hanced cell migration. Other growth factors should be tested for ef-fect in the presence of a soluble matrix proteoglycan and comparedto findings in the absence of the proteoglycan.

A much smaller number of growth factors were tested in thetransmigration studies using Boyden and transwell chambers andwere mostly chemotactic assays. It was noteworthy that TGF-b1at 2 ng/ml was effective in stimulating both chemokinetic and che-motactic migration. HGF at 6 ng/ml also enhanced chemokinetic


migration, but was less effective than TGF-b1 at 2 ng/ml. A combi-nation of HGF at 6 ng/ml and TGF-b1 at 2 ng/ml was more effectivethan either growth factor alone. FGF-2 at 1 ng/ml, FGF-7 at 10 ng/ml and AGF at 2 and 4 ng/ml stimulated chemotaxis. The transwellchamber assay and the scratch wound assay differ in that singlecells are used in the former and attached cells are used in the later.Nevertheless both assays showed a significant migration of kerat-inocytes in the presence of growth factors such as TGF-b1 and HGF.

In the assays of migration on ECM proteins, all the growth fac-tors and cytokines tested are known to stimulate wound healingin vivo. Migration of keratinocytes was followed for up to 24 hand under the conditions used it was considered that the assays as-sessed motility unconfounded by the possibility of cell division.EGF and TGF-a stimulated migration on type I collagen, type IV col-lagen and fibronectin, but there was very little or no migration onlaminin. Migration was enhanced on type IV collagen by IGF, andon type I collagen and type IV collagen by IL-1a. MGSA increasedmigration on fibronectin and laminin at low concentration (1 ng/ml) but not at higher concentration, and not on type IV collagen.All the other growth factors and cytokines tested did not enhancemigration on ECM proteins. This is surprising given that for exam-ple TGF-b1 enhances wound closure in rats [43], IL-6 augmentedepithelization and wound closure in the steroid-impaired healingof mice [44], and IL-8 enhanced epithelization of burns [45]. Inmany of these studies it might be that the extent of attachmentof keratinocytes to the ECM protein coating is responsible for a lackof stimulated migration. One study reported lamellipodia forma-tion being observed with FGF-2 at 1 ng/ml on type I collagen ma-trix [30].

The syndecan family of cell surface proteoglycans consists ofsingle transmembrane proteins containing conserved cytoplasmicand transmembrane domains and less well conserved extracellulardomains (ectodomains) which bear variable numbers of glycos-aminoglycan chains. Syndecans bind many of the factors thatorchestrate the inflammatory response to tissue injury as well asa variety of ECM components and adhesion molecules via theirheparan sulfate chains. Syndecan expression is highly regulatedduring wound repair. Also, syndecans on cell surfaces can becleaved near the plasma membrane to release the now soluble in-tact proteoglycan ectodomains into the surrounding milieu [46].This release is accelerated by activation of proteinases (e.g. throm-bin) and growth factor receptors (epidermal growth factor recep-tors) and by the direct action of proteinases involved in woundrepair (e.g. plasmin) [46]. Syndecan-1 and -4 ectodomains havebeen found in acute human dermal wound fluids. Glycosaminogly-cans may be regarded as occurring in a solid phase state (as inte-gral parts of proteoglycans at the cell surface, in the ECM), aswell as in the fluid phase (secreted by cells). Glycosaminoglycansmay have specific properties in regard to interactions with biolog-ical mediators depending on whether they are present in the solidor fluid phase.

4.5. Signaling pathways by which specific growth factors and cytokinesinfluence keratinocyte shape, migration and wound closure in vitro

Earlier studies have implicated several signaling pathways incutaneous wound healing such as signaling via mitogen-activatedprotein kinases (MAPKs), protein kinase C (PKC), and via the EGFreceptor (EGFR) [47–49]. Much of the signaling is directed at acti-vating migration into the wound site, but the mechanisms bywhich epidermal keratinocytes remodel their actomyosin cytoskel-eton to activate migration is not well understood. Myosin II has akey role in cell migration, in posterior cell retraction and helpingto stabilize focal adhesions formed in the anterior portion of thecell. Post-wounding, a rapid phosphorylation of myosin II occursand is far in advance of the later polarization and cell migration.

During this acute phase of myosin II activation, p38-MAPK andcytosolic calcium have critical roles driving cytoskeletal assembly[50].

In vivo, keratinocytes initiate migration from the wound edgeby extending lamellipodia into a provisional matrix of fibrin, fibro-nectin and vitronectin. Lamellipodia-like structures have beenfound in cultured keratinocytes treated with 4 ng/ml EGF. The sig-naling pathway that regulates lamellipodia formation involves gly-cogen synthase kinase-3 (GSK-3) activation. This was shown by thespecific GSK-3 inhibitor, SB-415286, which blocked lamellipodiaformation and EGF-induced migration of HaCaT keratinocytes inscratch wound assay when incubated with 30 lM SB-415286 be-fore the addition of 10 ng/ml EGF to the cells [51].

EGF and IGF-1 were shown to regulate keratinocyte motility byusing distinct signaling pathways. When subconfluent cultures ofprimary human keratinocytes were treated with 10 ng/ml EGF, atransient increase of MAPK phosphorylation and hence activationoccurred in cells after 15 and 30 min. This stimulation was sup-pressed when cells were preincubated with 10 lM of the specificMEK-1 inhibitor PD98059 [12]. Under the conditions employed,EGF did not stimulate activity of phosphoinositide 3-kinase (PI-3K)in keratinocytes using phosphorylated Akt (protein kinase B) as ameasure for PI-3K. By contrast, stimulation of subconfluent primaryhuman keratinocytes with 100 ng/ml IGF-1 resulted in pronouncedactivation of PI-3K but not of MAPK [12]. MAPK was shown to medi-ate EGF-stimulated keratinocyte motility. Incubation of keratino-cytes with 10 ng/ml EGF led to a 3-fold increase in cell motility. Inthe presence of 10 ng/ml EGF, inhibition of MAPK activity withPD98059 reduced keratinocyte motility [12]. Random cell migrationof primary human keratinocytes on fibronectin or collagen coateddishes was stimulated by 100 ng/ml IGF-1. Treatment with 25 lMLY294002, an inhibitor of PI-3K, reduced spontaneous keratinocytemovements in the absence of IGF-1 but did not reduce IGF-1-stimu-lated migration even when added at 50 lM concentration. It wasconcluded that PI-3K activity was not necessary for IGF-1-stimu-lated keratinocyte migration in vitro [12]. Activation of MAPK orPI-3K was shown to be sufficient to stimulate wound epithelizationusing confluent monolayers of keratinocytes [12].

HGF-stimulated migration of HaCaT keratinocytes was shownto occur via c-Met/PI-3K activated signaling. HGF caused rapidmigration of cells from the wound edge resulting in scratch woundhealing after 3 days of stimulation by 7% (25 ng/ml), 52% (100 ng/ml) and 62% (250 ng/ml). SU11274, a c-Met inhibitor, reducedHGF-induced (100 ng/ml) wound healing by 93% (0.85 lM) and97% (1.75 lM) [22]. HGF-linked downstream intracellular signal-ing in scratch wound healing involved the PI-3K pathway. Theselective inhibitors of PI-3K, wortmannin (1 lM) and LY294002(10 lM), decreased HGF-induced (100 ng/ml) wound healing by102% and 83%, respectively [22].

A study was performed to determine whether growth factor-in-duced migration of keratinocytes was dependent on the inductionof EGFR signaling by endogenously expressed HB-EGF. The concen-trations of EGF and TGF-a that achieved wound closure of scratch-wounded HaCaT cell monolayers in 24 h were 2 ng/ml and 3 ng/ml,respectively [24]. Neither TGF-b1 or FGF-7 was able to cause com-plete wound closure in 24 h; maximum migration was obtainedwith 10 ng/ml TGF-b1 and 2 ng/ml FGF-7. Except for FGF-7, thegrowth factors did not increase cell numbers beyond control levelsduring the 24-h migration. Incorporation of 4 mM hydroxyurea didnot alter the migration results. Function blocking antibodiesagainst EGFR and HB-EGF caused a significant decrease in cellmigration regardless of the stimulating growth factor, whereasthe EGFR tyrosine kinase inhibitor typhostin AG1478 completelyblocked migration. Moreover, GSK-3 inhibitor SB-415286 potentlyblocked cell migration induced by all of these growth factors[24]. Scratch wound assays with primary human keratinocytes


produced similar results. It was concluded that GSK-3 and EGFRsignaling, together with autocrine HB-EGF expression, are potentmodulators of keratinocyte migration [24].

Functionally blocking the EGF/TGF-a receptor with anti-EGFRantibody did not influence the IL-1a-driven migration on type Ior type IV collagen [36]. This together with delayed onset of migra-tion compared with that seen with EGF/TGF-a and the absence ofthe characteristic up-regulation of a2 integrin subunit seen withEGF/TGF-a stimulation confirmed the presence of an effector path-way on migration for IL-1a distinct from that for EGF/TGF-a anddoes not involve changes in the expression of integrin subunits[36].

The p38-MAPK pathway was shown to be required for keratino-cyte migration on type I collagen as evidenced by the inhibitors SB-202190, SB-203580, rottlerin, and PPI potently blocking migrationwithout compromising cell viability [52]. It was suggested that todrive keratinocyte migration the p38-MAPK pathway needs towork together with other parallel signaling pathways [52]. Furtherstudies are required for a full understanding of the ultimate signal-ing mechanisms in the control of keratinocyte migration as influ-enced by growth factors and cytokines and the environment ofthe cells which includes ECM proteins.

5. Conclusion

Three methods of assaying keratinocyte migration have beenreviewed in this paper. The scratch wound assay uses cells in amonolayer attached to the surface of culture dishes or wells. Thetransmigration assay with Boyden or transwell chambers usesunattached cells. The assay in the presence of ECM proteins usescells attached to a protein coating on the coverslip or dish. Allthe growth factors tested in the scratch assay are known to stimu-late wound healing in animal models, and all were shown to en-hance keratinocyte migration into the cell-free area made in themonolayer. Only a small number of growth factors have beentested in the transmigration assay, but several notable findingshave been observed. TGF-b1 and HGF are effective in inducing che-mokinetic migration in the scratch wound and chamber assay, andin both assays the combination of these two growth factors is morestimulatory than either alone. This combination of growth factorshas not yet been tested to determine whether it can accelerate ree-pithelization and wound healing in experimental animals. Also ofinterest is the combination of IGF-1 and EGF which had a greaterstimulatory effect in the scratch wound assay than either growthfactor alone. FGF-7 at a suitable concentration was shown to bechemotactic for keratinocytes. The findings regarding migrationon immobilized type I collagen, type IV collagen, fibronectin andlaminin indicated that only a few of the growth factors and cyto-kines tested enhanced migration. EGF, TGF-a and IGF-1 enhancedmigration in scratch wound assay and also on some of these ECMproteins. The ECM functions to regulate attachment, proliferation,migration, and differentiation and survival of cells. Also growthfactors can be firmly associated with the ECM. For example, FGF-2 is bound to heparan sulfate in the ECM, and members of theEGF family also bind to heparan sulfate proteoglycan. If the growthfactor binds to the immobilized ECM protein molecules on the cov-erslips or dishes, then this may affect the way in which the growthfactor influences keratinocyte migration. It is noteworthy that FGF-10 in combination with soluble dermatan sulfate stimulatedmigration of NHEK cells. Further studies are needed to examinethe influence of other ECM proteoglycans (such as heparan sulfate,hyaluronic acid) in the fluid phase together with selected growthfactors on keratinocyte migration in vitro.

The scratch wound assay provides a useful, inexpensive andeasy-to-perform screening method for testing individual or combi-

nations of growth factors or cytokines prior to performing woundhealing studies with laboratory animals. Combinations of growthfactors that might be screened for effect are FGF-2 and TGF-b1,HGF and TGF-b1, PDGF and EGF, PDGF and TGF-a, PDGF and IGF-1, FGF-2 and IGF-2. This would examine whether the effect onkeratinocyte migration in the scratch assay is a predictor of en-hanced wound closure in vivo. Treatment with the combinationPDGF and IGF-1, PDGF and IGF-II, or PDGF and TGF-a has beenshown to be a powerful stimulator of wound healing in vivo[53–55]. Also the scratch wound assay enables the effect of growthfactor treatment combined with other modalities, such as laserirradiation, to be tested before proceeding to examine their effecton wound healing in experimental animals.

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a review of the influence of growth factors and cytokines in in vitro human keratinocyte migration

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