Effects of Substrate-Coating Materials on the Wound-Healing Process

materials

Article Eﬀects of Substrate-Coating Materials on the Wound-Healing Process

Jin-Young Lin 1, Kai-Yin Lo 2 and Yung-Shin Sun 1,* 1 Department of Physics, Fu-Jen Catholic University, New Taipei City 24205, Taiwan 2 Department of Agricultural Chemistry, National Taiwan University, Taipei 10617, Taiwan * Correspondence: [email protected]; Tel.: +886-2-2905-2585

Received: 25 July 2019; Accepted: 27 August 2019; Published: 29 August 2019

Abstract: The wound-healing assay is commonly and widely used for investigating collective cell migration under various physical and chemical stimuli. Substrate-coating materials are shown to affect the wound-healing process in a cell-type dependent manner. However, experiment-to-experiment variations make it difficult to compare results from different assays. In this paper, a modified barrier wound-healing assay was reported for studying the wound-healing process on different substrates in one single petri dish. In short, half of a dish was covered with the tape, and coating materials, poly-l-lysine and gelatin, were applied to the surface. After peeling off the tape, half of the surface was coated with the desired material. Then a customized barrier was placed inside the dish to create the wound. The results indicated that surface coating did not affect cell proliferation/viability, and the wound-healing rate increased in coated surfaces compared to uncoated ones. The present study provides a platform for further understanding the mechanisms of substrate coating-dependent wound-healing processes.

Keywords: wound-healing assay; cell migration; wound-healing rate; ﬁbroblasts; surface coating

1. Introduction Cell migration is directed by various chemical and physical cues, phenomena named “-taxis”. Chemotaxis, the directional migration of somatic cells in response to certain chemical gradient, plays important roles in many physiological processes such as cancer metastasis [1,2] and cell development [3,4]. Electrotaxis, characterizing how adherent cells move under the stimulus of direct current or alternating current electric fields, has been reported to be crucial in wound healing [5,6] and nerve regeneration [7]. Other types of “-taxis” have been identified and investigated, including phototaxis (stimulated by a light gradient) [8], aerotaxis (stimulated by an oxygen gradient) [9], thermotaxis (stimulated by a temperature gradient) [10], durotaxis (stimulated by a stiffness gradient) [11], and magnetotaxis (stimulated by a magnetic field gradient) [12]. Induced by one or more of above-mentioned stimuli, cells can migrate collectively as a consequence of cell–cell communication and cell-environment interactions. Cells usually form the so-called self-assembled monolayers where they are attached to each other in biochemical and/or mechanical ways. To follow the dynamic process of collective cell migration as well as better understand its underlying mechanisms, researchers have developed various in vitro techniques, including wound-healing assays [13,14]. In a wound-healing assay, a cell-free region is created from a cell monolayer. Cells migrate to cover the wound, and time-lapse images are recorded using a microscope. Then based on these images, wound-healing rates can be calculated. The most common method for creating a wound is to scratch out a cell-free area using a tip or needle [15–17]. This scratch wound-healing assay has been commercialized as the CellPlayer Migration Assay by Essen BioScience (Ann Arbor, MI, USA) [18]. The advantages of this method include (1) it is easily conducted and quick, and (2) it can be applied

Materials 2019, 12, 2775; doi:10.3390/ma12172775 www.mdpi.com/journal/materials Materials 2019, 12, 2775 2 of 10 on any substrates. However, using tips to mechanically create the wound could do damage to the cells and the cell culture surface. It is also difficult to control the size and shape of the wound from experiment to experiment. All these make the results unreliable and hard to be compared. Instead, by placing a stopper onto the substrate prior to seeding cells and waiting until a cell monolayer is formed, a cell-free region is created after removing the barrier [13,19]. In this barrier wound-healing assay, the cells and the surface remain intact, and the size and shape of the wound can be precisely controlled. Platypus Technologies (Madison, WI, USA) and Ibidi (Martinsried, Germany) have commercialized this assay by placing stoppers inside each well of a 96-well plate to increase the experimental throughput. As substitutes, various wound-healing assays have been developed, including those based on laser and ultraviolet light ablation [20,21], current-induced cell electroporation [22], and trypsin-caused cell dissociation in microfluidic devices [23,24]. The wound-healing process, quantified as the healing rate, is affected by a number of factors, such as cell type, cell culture substrate (coating and stiffness), and other physical /chemical stimuli. It was found that, using fibroblast cell line NIH 3T3 as the model, both cell culture serum and electric field increased the wound-healing rate obviously [5]. And in the same cell model, chemicals β-lapachone and honokiol were shown to increase and decrease the wound-healing rates, respectively [5,21]. As far as the cell culture surface is concerned, skin explants cultured on 2–50 kPa collagen-coated substrates rapidly re-epithelialized within 10–15 h, but in harder (1 GPa) and other coatings (tenascin, fibronectin, and laminin), the wound recovered slowly [25]. The wound-healing rate depends on not only the substrate-coating material but also on the cell type. Klein et al. reported that fibronectin accelerated the collective migration of Schwann cells, but collagen I, laminin, poly-l-lysine, and poly-l-ornithine showed little or no migration-promoting effects [26]. However, another research using prostate cancer cells as the model exhibited conflicting results. It was observed that cells grown in the presence of laminin migrated 62% faster than the control cells, but poly-l-lysine, poly-l-ornithine, and fibronectin caused cells to migrate slower than the control, displaying reduced wound-healing rates of 15%, 33%, and 20%, respectively [27]. In these two studies, the scratch wound-healing assays were used, which could damage the coatings and possibly cause further experiment-to-experiment variations. To get rid of the drawbacks of the scratching method, here we report a modified barrier wound-healing assay for investigating collective cell migration on different coated substrates. Fibroblasts are commonly used as the wound-healing model because they play critical roles in processes of breaking down the fibrin clot, creating new extra cellular matrix, and contracting the wound [28–30]. One half of a culture dish was coated with poly-l-lysine or gelatin, and the other half was uncoated. Prior to seeding cells, a barrier was placed onto the substrate. After a cell monolayer was formed, the stopper was removed to form a wound. The wound-healing processes under coated and uncoated surfaces were followed simultaneously to obtain the healing rates for comparison. This design greatly eliminated the possibilities of damages to the coatings and run-to-run variations. The present study provides a platform for accurate and unbiased investigation of substrate coating-dependent wound-healing processes.

2. Materials and Methods

2.1. Cell Culture and Chemicals The mouse embryonic fibroblast cell line NIH 3T3 was purchased from the Bioresource Collection and Research Center (BCRC), Hsinchu, Taiwan. A complete medium consisting of Dulbecco’s modified eagle medium (DMEM, Gibco, Waltham, MA, USA) and 10% calf serum (CS, Invitrogen, Carlsbad, CA, USA) was used for cell culture. The cells were incubated in tissue culture polystyrene flasks (Corning, Corning, NY, USA) in 5% CO at 37 C until a density of 5~6 104 cells/cm2 was reached. 2 ◦ × For surface coating, gelatin (Sigma, St. Louis, MO, USA) and poly-l-lysine (Sigma, St. Louis, MO, USA) were diluted in 1 phosphate-buffered saline (PBS) into concentrations of 0.1% (w/w) and 0.1 µg/mL, × respectively, as recommended by the manufacturer. Coomassie Brilliant Blue (Bionovas, Toronto, ON, Materials 2019, 12, x FOR PEER REVIEW 3 of 10

Materials 2019, 12, 2775 3 of 10 Brilliant Blue (Bionovas, Toronto, ON, Canada) was diluted into a concentration of 2.5 g/L (in 0.1 L acetic acid, 0.3 L methanol, and 0.6 L ultrapure water) for verifying the presence of gelatin and Canada)poly-L-lysine. was diluted into a concentration of 2.5 g/L (in 0.1 L acetic acid, 0.3 L methanol, and 0.6 L ultrapure water) for verifying the presence of gelatin and poly-l-lysine. 2.2. Cell Viability Assay 2.2. Cell Viability Assay The proliferation and viability of fibroblasts were quantified using the MTT (3-(4,5The-Dimethylthiazol proliferation and-2- viabilityyl)-2,5-diphenyltetrazolium of ﬁbroblasts were quantiﬁed bromide) using assay the MTT (Sigma). (3-(4,5-Dimethylthiazol- This yellow 2-yl)-2,5-diphenyltetrazoliumtetrazolium salt was transformed bromide) into assay purple (Sigma). formazan This yellow crystal tetrazolium by live cells. salt Fibroblasts was transformed were intopassag purpleed to formazan a 12-well crystal plate dividedby live cells. into Fibroblasts three groups: were Control passaged (uncoated), to a 12-well PLL plate (coated divided with into 0.1 threeμg/mL groups: poly-L Control-lysine), (uncoated), and Gelatin PLL (coated (coated wit withh 0.1% 0.1 µ gelatin).g/mL poly- Afterl-lysine), 24 h, 400 and μL Gelatin of MTT (coated solution with 0.1%(0.5 mg/ml gelatin). in After DMEM) 24h, was 400 addedµL of MTT to each solution well (0.5 for incubationmg/ml in DMEM) at 37 °C was for added 2 h. Then to each 400 well μL for of incubationsolubilization at 37solution,◦C for 2dimethyl h. Then 400sulfoxideµL of solubilization(DMSO), was solution,added to dimethyl the wells sulfoxide for 5 min (DMSO), incubation. was addedThe absorbance to the wells w foras measured 5 min incubation. using an The ELISA absorbance (enzyme was-linked measured immunosorbent using an ELISA assay (enzyme-linked) plate reader immunosorbent(Tecan, Männedorf assay), Switzerland plate reader) at (Tecan, 570 nm. Männedorf, Two independent Switzerland) experiments at 570 nm. were Two performed independent on experimentsseparate plates. were performed on separate plates.

2.3. Fabrication of Barrier The I-shapedI-shaped barrier was made of polydimethylsiloxanepolydimethylsiloxane (PDMS, Dow Corning Sylgard 184184,, Midland,Midland, MI,MI, USA).USA). First, a negative polymethylmethacrylate (PMMA) mold was fabricated using a CO laser scriber (MS640D, Ming-Cheng Technics Corp., Nantou, Taiwan). A number of these molds CO2 laser scriber (MS640D, Ming-Cheng Technics Corp., Nantou, Taiwan). A number of these weremolds put were inside put a inside petri dish a petri (diameter dish (diameter= 10 cm, SPL= 10 Lifecm, Sciences,SPL Life Gyeonggi-do,Sciences, Gyeonggi Korea),-do, and Korea the PDMS), and solutionthe PDMS (1/ 10,solutionw/w curing (1/10, agent w/w tocuring prepolymer) agent to was prepolymer) poured into was the dishpoured to cover into the molds.dish to The cover molds the weremolds. put The under molds vacuum were put (~60 under Torr) vacuum for 30 min(~60 and Torr then) for baked30 minat and 50 then◦C for baked 4 h andat 50 then °C for left 4 underh and sterilethen left conditions under sterile overnight. conditions The I-shapedovernight. PDMS The I barriers-shaped werePDMS cut barriers oﬀ and were removed cut off from and the removed dish in preparationfrom the dish for in use preparation in experiments for use (see in experiments Figure1). (see Figure 1).

FigureFigure 1.1. Picture of the customized PDMSPDMS I-shapedI-shaped barrier.barrier. ScaleScale barbar == 1.5 cm.

2.4. Experimental Procedure The experimentalexperimental procedure procedure is is illustrated illustrated in in Figure Figure2 and 2 and described described as follows. as follows. First, First, half ofhalf a petri of a dishpetri (diameterdish (diameter= 3.5 cm,= 3.5 TrueLine, cm, TrueLine Rochester,, Rochester, NY, USA) NY, wasUSA blocked) was blocked with the with tape the (thickness tape (thicknes= 60 µsm =, 8018,60 μm, 3 M)8018, (Figure 3 M)2 a).(Figure The solution,2a). The washingsolution, buffer washing (PBS, buffer for Control, (PBS, for Sigma, Control St. Louis,, Sigma, MO, St. USA),Louis, 0.1%MO, gelatin,USA), 0.1% or 0.1 gelatin,µg/mL or poly- 0.1 μg/mLl-lysine, poly was-L poured-lysine, intowas thepoured dish in (Figureto the2 dishb). After (Figure 30 2b min,). After the solution 30 min, wasthe solution removed, was and removed, the dish and was the washed dish withwas washed PBS a few with times PBS and a few then times dried and (Figure then 2driedc). The (Figu tapere was2c). The removed tape (Figurewas removed2d) and (Figure cell medium 2d) and (DMEM cell medium+ 10% (DMEM CS) was poured+ 10% CS) into was the dishpoured (Figure into 2thee). Thedish I-shaped(Figure 2e barrier). The I was-shaped placed barrier in the was middle placed of in the the dish middle (Figure of the2f) dish and then(Figure2.5 2f) 10and5 cells then were2.5 × × seeded105 cells inside were (Figureseeded 2insideg). After (Figure a cell 2g monolayer). After a cell was monolayer grown (Figure was 2grownh), the (Figure barrier 2h was), the removed barrier (Figurewas removed2i). Figure (Figure2j shows 2i). Figure the top 2 viewj shows of the dish,top view where of the upperdish, where part was the coatedupper part (blue), was and coated the lower(blue), part and wasthe lower uncoated part (white). was uncoated (white).

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Figure 2. Experimental procedure. (a)–(i) Side view of the dish. (a) Block half of the dish with the tape. (b) Pour in the solution. (c) Wait until dried. (d) Remove the tape. (e) Add in the culture Figure 2. Experimental procedure.procedure. ((aa–)i–)(i Side) Side view view of theof the dish. dish. (a) ( Blocka) Block half half of the of dishthe dish with with the tape. the medium. (f) Place the stopper in the middle of the dish. (g) Seed in cells. (h) Wait until a cell (tape.b) Pour (b) in Pour the solution.in the solution. (c) Wait (c until) Wait dried. until (d dried.) Remove (d) Remove the tape. the (e ) tape. Add (ine) the Add culture in the medium. culture monolayer was formed. (i) Remove the stopper. (j) Top view of the dish. Green: tape. Blue: washing (medium.f) Place the (f) stopper Place the in the stopper middle in of the the middle dish. ( ofg) theSeed dish. in cells. (g) Seed(h) Wait in cells.until a (h cell) Wait monolayer until a was cell buffer (PBS). Red: Cell medium (DMEM + 10% CS). Light gray: stopper. Dark gray: cell. monolayerformed. (i) Removewas formed. the stopper. (i) Remove (j) Top the view stopper. of the (j dish.) Top Green:view of tape. the dish. Blue: Green: washing tape. bu ffBlue:er (PBS). washing Red: bufferCell medium (PBS). Red: (DMEM Cell+ medium10% CS). (DMEM Light gray: + 10% stopper. CS). Light Dark gray: gray: stopper. cell. Dark gray: cell. 2.5. Data Analysis 2.5. Data Analysis 2.5. DataThe Analysis wound- healing process was recorded using a bright-field inverted microscope (ESPA, Hsinchu,The wound-healing woundTaiwan).-healing Seven process images process was (see was recorded Figure recorded 3a using) were using a bright-field taken a brigh at 0,t inverted- field12, 24, inverted 36, microscope and 48 microscope h(ESPA, after removing Hsinchu, (ESPA, Taiwan).Hsinchu,the barrier. SevenTaiwan). They images Seven were (see images further Figure (see3 analyzeda) Figure were taken using3a) were at ImageJ,0, 12,taken 24, at which36, 0, and 12, is 4824, a h36, after free and removing Java 48 h-based after the removing software barrier. Theythedeveloped barrier. were by further They the National analyzedwere further Insti usingtutes analyzed ImageJ, of Health which using (Version is ImageJ, a free1.50c Java-based which, NIH, Bethesda, is a software free MD Java developed, -USAbased). As software shown by the Nationaldevelopedin Figure Institutes3b by, this the Nationalsoftware of Health Instiwas (Version tutesused ofto 1.50c, Healthdraw NIH, the (Version boundaries Bethesda, 1.50c MD, ,of NIH, the USA). Bethesda,wound As shownat MDdifferent, inUSA Figure time). As3 points.b,shown this softwareinThe Figure areawas 3b enclosed, this used software to by draw these thewas boundaries boundariesused to draw of could the the wound boundaries be calculated. at diff erentof the And time wound points.the at wound different The area-healing time enclosed points. rate by is (푅푖−푅푓) (Ri R f ) theseThecalculated area boundaries enclosed as could by× 100% be these calculated. , where boundaries R Andi and could the R wound-healingf are be calculated. the initial rate And an isd calculatedfinal the wound area ass - ofhealing − the wound, rate100%, is 푅푖 Ri (푅푖−푅푓) × wherecalculatedrespectively.Ri and as For Rf are each the× condition,100% initial, andwhere three final R independenti areasand ofRf theare wound,runs the were initial respectively. performed. and final For Therefore, area eachs condition, of there the wound,were three a 푅푖 independentrespectively.total of nine (three runsFor each were images condition, performed. in each three of Therefore, three independent runs) there data were runspoints a were total to be ofperformed. analyzed nine (three toTherefore, images get the instandard there each ofwere threeerror a runs)totalof the of datamean nine points (SEM).(three to images be analyzed in each to of get three the standardruns) data error points of theto be mean analyzed (SEM). to get the standard error of the mean (SEM).

Figure 3.3. (a) SevenSeven images were taken at oneone timetime point.point. The upper three images were used for the Figurecoated substrate.substrate.3. (a) Seve TheThen images lowerlower were threethree taken images images at were wereone usedtime used forpoint. for the the The uncoated uncoated upper substrate. three substrate. images (b ()b ImageJ )were ImageJ used was was usedfor used the to coateddrawto draw the substrate. the boundaries boundaries The andlower and calculate threecalculate images the the area areawere of theof used the wound. wound.for the uncoated substrate. (b) ImageJ was used to draw the boundaries and calculate the area of the wound. 3. Results and Discussion 3. Results and Discussion

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3. Results and Discussion

Materials 2019, 12, x FOR PEER REVIEW 5 of 10 3.1. Verifying the Presence of Coating Materials 3.1.Coomassie Verifying Brilliantthe Presence Blue of Coating is commonly Materials and widely used for staining proteins. After blocking half of the petriCoomassie dishes with Brilliant the Blue tapes is (seecommonly Figure and4a), widely solutions used of for PBS staining (C, for proteins. control), After poly-lysine blocking (P), and gelatinhalf of the (G) petri were dishes added with into the tapes dishes. (see Then Figure solutions 4a), solutions were removed, of PBS (C, the for dishes control), were poly washed-lysinewith PBS,(P), and and the gelatintapes were (G) were removed. added Stained into the with dishes. Coomassie Then solutions Brilliant were Blue removed, for 30 min the and dishes washed were with ultrapurewashed water with a PBS, few and times, the the tapes dishes were were removed. photographed Stained with and Coomassie are shown Brilliant in Figure Blue4b–d. for 30 As min clearly seen,and surfaces washed coated with withultrapure poly- waterl-lysine a few (Figure times,4 c the left, dishes marked were with pho atographed circle) and and gelatin are shown (Figure in 4d Figure 4b–d. As clearly seen, surfaces coated with poly-L-lysine (Figure 4c left, marked with a circle) left, marked with a circle) were much bluer than those that were uncoated (Figure4b–d right, marked and gelatin (Figure 4d left, marked with a circle) were much bluer than those that were uncoated with crosses) and coated with PBS (Figure4b left, marked with a circle). Therefore, it was verified that (Figure 4b–d right, marked with crosses) and coated with PBS (Figure 4b left, marked with a circle). l poly-Therefore,-lysine and it was gelatin verified were that successfully poly-L-lysine coated and ongelatin the surfaces. were successfully The concentrations coated on the used surfaces. here were recommendedThe concentrations by the manufacturer used here were for surfacerecommended modification. by the manufacturer Different dilutions for surface were modifalso tried,ication. but no betterDifferent results dilutions were obtained were also (data tried, not but shown). no better results were obtained (data not shown).

FigureFigure 4. 4.( a(a)) HalfHalf of of the the petri petri dishes dishes were blocked were blocked with the tapes. with Pictures the tapes. of Coomassie Pictures Brilliant of Coomassie Blue Brilliantstaining Blue ofstaining (b) phosphate of (b)-buffered phosphate-bu saline ﬀ (PBSered)-coated saline (C), (PBS)-coated (c) poly-L- (C),lysine (c-coated) poly- (P),l-lysine-coated and (d) (P), andgelatin (d)-coated gelatin-coated dishes (G). dishes Circle: (G). coated Circle: half. coated Cross: uncoated half. Cross: half. uncoated half.

3.2. Cell3.2. Cell Morphology Morphology and and Proliferation Proliferation/Viability/Viability FigureFigure5a shows 5a shows the the images images of of cells cells grown grown on on didifferentfferent substrate substratess after after 24 24h. h.Without Without detailed detailed analysis, the sizes and shapes of cells on uncoated and coated surfaces were similar, suggesting that analysis, the sizes and shapes of cells on uncoated and coated surfaces were similar, suggesting that these these coatings did not alter cell morphology. The result of the 24 h MTT assay is shown in Figure 5b, coatings did not alter cell morphology. The result of the 24 h MTT assay is shown in Figure5b, where where the absorbance was normalized to that of the control (uncoated) group. As indicated, the the absorbancenormalized was values normalized were 97.9% to that and of the 105.7% control for (uncoated) poly-L-lysine group.- and As gelatin indicated,-coated the surfaces, normalized valuesrespectively. were 97.9% Compared and 105.7% with for the poly- uncoatedl-lysine- surface, and these gelatin-coated two coatings surfaces, slightly respectively.reduce and increase Compared withthe the proliferation/ uncoated surface,viability these of fibroblasts, two coatings respectively. slightly Student’s reduce and t-tests increase were performed the proliferation, showing/viability no of fibroblasts,statistically respectively. significant differences. Student’s Therefore,t-tests were it was performed, suggested showing that theseno two statistically materials did significant not differences.significantly Therefore, affect cell it was proliferation/viability. suggested that theseIt was two reported materials that did Schwann notsignificantly cells cultivated aff ect on cell proliferationpoly-L-lysine/viability. showed It nowas difference reported in that cell Schwann viability when cells cultivated compared with onpoly- the cellsl-lysine cultured showed on no differenceuncoated in polystyrene cell viability after when two days compared [26]. Human with thecolon cells adenocarcinoma cultured on cells uncoated also exhibited polystyrene similar after twoproliferation days [26]. Humanrates on gelatin colon- adenocarcinomacoated and uncoated cells microflui also exhibiteddic channels similar after two proliferation days of culture rates on [31]. gelatin-coated and uncoated microfluidic channels after two days of culture [31].

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FigureFigure 5. 5. (a(a) )Cells Cells grown grown on on uncoated uncoated (left), (left), poly poly--Ll-lysine-lysine-coated-coated (middle), (middle), and and gelatin gelatin-coated-coated (right) (right) surfacessurfaces after after 24 24 h. h. Scale Scale bar bar == 100100 μm.µm. (b ()b Cell) Cell proliferation/viability proliferation/viability on on different different surfaces surfaces after after 24 24 h h.. Control:Control: uncoated. uncoated. PLL: PLL: Poly Poly--Ll-lysine.-lysine. Statistical Statistical analysis analysis was was performed performed on on eight eight independent independent data data pointspoints (see (see Section Section 2.2). 2.2). Student’s Student’s t-testst-tests were were performed. performed. ns:ns: no nostatistically statistically significant significant difference difference (p >( p0.05)> 0.05)..

3.3. Dependence of Substrate-Coating Materials on the Wound-Healing Process 3.3. Dependence of Substrate-Coating Materials on the Wound-Healing Process The wound-healing process was followed by taking time-lapse images at 0, 12, 24, 36, and 48 h The wound-healing process was followed by taking time-lapse images at 0, 12, 24, 36, and 48 h afterafter removing removing the the barrier. barrier. Figure Figure 66 shows a representative image taken in the boundary between gelatin-coated and uncoated surfaces. The width of the wound was around 1.5 mm, equal to that gelatin-coated and uncoated surfaces. The width of the wound was around 1.5 mm, equal to that of theof theI-shaped I-shaped barrier. barrier. After After 48 h 48, the h, thewound wound in the in thecoated coated surface surface was was nearly nearly closed closed compared compared to to that in the uncoated surface. Figure7 shows the images of the wounds at di fferent time points. that in the uncoated surface. Figure 7 shows the images of the wounds at different time points. As l obviouslyAs obviously indicated, indicated, cells cells migra migratedted faster faster on onpoly poly--L-lysine-lysine-- (Figure (Figure 7b7b Coate Coated)d) and and gelatin gelatin-coated-coated (Figure7c Coated) surfaces than on PBS-coated (Figure7a Coated) and uncoated surfaces (Figure7a–c) (Figure 7c Coated) surfaces than on PBS-coated (Figure 7a Coated) and uncoated surfaces (Figure 7aNon-coated).–c) Non-coated). There There were twowere types two oftypes control of control conditions. conditions. In one, In half one, of thehalf dish of the was dish blocked, was b meaninglocked, meaningthat this that part this was part uncoated. was uncoated. In the other, In the PBS other, was PBS used was as used the coating as the coating material, material, suggesting suggesting that the thatentire the dish entire was dish uncoated. was uncoated. Comparing Comparing the coated the andcoated non-coated and non- imagescoated images in Figure in7 Figurea, it was 7a implied, it was impliedthat the residues that the after residues removing after the removing tapes (if there the were tapes any) (if did there not were affect the any) wound-healing did not affect process. the woundQuantitatively,-healing process. the wounding Quantitatively, areas at dithefferent wounding time pointsareas at were different measured time andpoints the were corresponding measured t andwound-healing the corresponding rates were wound calculated-healing and rates shown were in Figure calculated8a. Student’s and shown-tests in were Figure performed 8a. Student’s on all l ttime-tests points. were Bothperformed gelatin- on and all poly- time-lysine-coated points. Both surfaces gelatin accelerated- and poly the-L wound-healing-lysine-coated processes surfaces acceleratedcompared with the wound uncoated-healing ones. processes For gelatin, compared the healing with rates uncoated were 74%, ones. 53%, For 30%, gelatin, and the 13% healing at 48 h, rates36 h, were 24 h, 74%, and 1253%, h, respectively.30%, and 13% These at 48 values h, 36 wereh, 24 89%,h, and 60%, 12 h 57%,, respectively. and 32%higher These thanvalues those were of p 89%,uncoated 60%, surfaces.57%, and As32% shown higher in than Figure those8b, of the uncoated-value surfaces. between gelatin-coatedAs shown in Figure and uncoated 8b, the p groups-value l betweenat 48 h was gelatin less-coated than 0.0001. and uncoated For poly- groups-lysine, at the 48 h healing was less rates than were 0.0001. 58%, For 40%, poly 24%,-L- andlysine, 11% the at healing48 h, 36 rates h, 24 were h, and 58%, 12 h, 40%, respectively. 24%, and Similarly,11% at 48 these h, 36 values h, 24 wereh, and 49%, 12 54%,h, respectively. 60%, and57% Similarly, higher l thesethan thosevalues of were uncoated 49%, 54%, surfaces. 60%, As and shown 57% higher in Figure than8c, those the p -valueof uncoated between surfaces. poly- -lysine-coatedAs shown in Figureand uncoated 8c, the p groups-value at between 48 h was poly less-L- thanlysine 0.001.-coated In and the controluncoated (PBS) groups group, at 48 the h healing was less rates than in 0.001.the PBS-coated In the control surface (PBS) were group close, the to healing those in rates the uncoatedin the PBS surface,-coated withsurface errors were of close 0.24%, to 1.7%,those 3%,in theand uncoated 10% at 48 surface, h, 36 h, 24 with h, and errors 12 h, of respectively. 0.24%, 1.7%, The 3%,p-value and between 10% at 48 these h, two 36 h groups, 24 h, indicated and 12 h no, respectively.statistically significant The p-value diff betweenerence as these shown two in Figuregroups8 d.indicated This again no verifiedstatistically that significant the tape did diff noterence a ffect asthe shown wound-healing in Figure 8d rate.. This again verified that the tape did not affect the wound-healing rate. Gelatin,Gelatin, an an irreversibly irreversibly hydrolyzed hydrolyzed form form of of collagen, collagen, is is widely widely used used as as a a coating coating material material in in cellcell culture culture for for improving improving cell cell attachment attachment [32] [32].. It It was was reported reported that that smooth smooth muscle muscle cells cells migrated migrated fasterfaster on on the the gelatin gelatin-coated-coated surface surface than than on on the the uncoated uncoated polystyrene polystyrene surface surface [33] [33].. An An increase increase of of 63% in the migration rate from 24 to 48 h was observed in a barrier wound-healing assay [33].

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Materials 2019, 12, x FOR PEER REVIEW 7 of 10 63% in the migration rate from 24 to 48 h was observed in a barrier wound-healing assay [33]. Under the same conditions, for human umbilical vein endothelial cells, the migration rate was even six Under the same conditions, for human umbilical vein endothelial cells, the migration rate was even times faster on the gelatin-coated surface than on the uncoated polystyrene one [33]. Poly-l-lysine six timesMaterials faster2019, 12 , x on FOR the PEER gelatinREVIEW -coated surface than on the uncoated polystyrene 7 one of 10 [33]. is a synthetic, positively charged amino acid chain commonly used as a coating material to enhance Poly-L-lysine is a synthetic, positively charged amino acid chain commonly used as a coating cell adhesionUnder the same [34,35 conditions,]. In the presentfor human study, umbilical poly- veinl-lysine endothelial was showncells, the to migration accelerate rate wound was even healing material to enhance cell adhesion [34,35]. In the present study, poly-L-lysine was shown to in fibroblasts.six times faster Somaiah on et the al. gelatin found-coated that, by surface using than the scratching on the uncoated method, polystyrene mesenchymal one stem[33]. cells accelerate wound healing in fibroblasts. Somaiah et al. found that, by using the scratching method, migratedPoly-L faster-lysine on is the a synthetic, poly-l-lysine positively surface charged (with anamino average acid migration chain commonly speed used of 6 µ asm / ah) coating than on the mesenchymalmaterial to stem enhance cells cell migrated adhesion faster [34,35] on. the In thepoly present-L-lysine study, surface poly (with-L-lysine an was average shown migration to uncoated one (with an average migration speed of 3.6 µm/h) [36]. However, contrary results were speedaccelerate of 6 μm/ woundh) than healing on the in fibroblasts. uncoated Somaiah one (with et al. an found average that, migration by using the speed scratching of 3.6 method, μm/h) [36]. reported on different cell lines. In a scratch wound-healing assay using Schwann cells as the model, However,mesenchymal contrary stem results cells were migrated reported faster on thedifferent poly-L -celllysine lines. surface In a (withscratch an averagewound - migrationhealing assay the wounding area after 48 h was still 67.5% of the original one on the poly-l-lysine coated surface, usingspeed Schwann of 6 μm/cellsh) as than the on model, the uncoated the wounding one (with area an averageafter 48 migrationh was still speed 67.5% of 3.6of the μm/ originalh) [36]. one compared to 54.4% on the uncoated surface [26]. Results from another scratch assay indicated that on theHowever, poly-L -contrarylysine coated results surface, were reported compared on different to 54.4% cell on lines. the Inuncoated a scratch surface wound -[26]healing. Results assay from poly-usingl-lysine Schwann caused cells prostate as the cancermodel, cellsthe wounding to migrate area slower after than48 h was the control,still 67.5% displaying of the original a reduction one of another scratch assay indicated that poly-L-lysine caused prostate cancer cells to migrate slower woundon the density poly-L by-lysine 15% coated [27]. Itsurface, is therefore compared suggested to 54.4% that on the the uncoated effect of surface poly-l -lysine[26]. Results coating from on the than the control, displaying a reduction of wound density by 15% [27]. It is therefore suggested that wound-healinganother scratch process assay is indicated cell type-dependent. that poly-L-lysine caused prostate cancer cells to migrate slower the effectthan theof polycontrol,-L-lysine displaying coating a reduction on the woundof wound-healing density process by 15% [27]is cell. It typeis therefore-dependent. suggested that the effect of poly-L-lysine coating on the wound-healing process is cell type-dependent.

Figure 6. A representative image showing the wound-healing process in gelatin-coated and FigureFigure 6. 6. AA representative representative image image showing showing the wound-healing the wound-healing process process in gelatin-coated in gelatin and-coated uncoated and uncoated surfaces. (a) The wound at time = 0 h. (b) The wound at time = 48 h. Scale bar = 500 μm. uncoatedsurfaces. surfaces. (a) The wound (a) The at wound time = at0 h.time (b) = The 0 h. wound (b) The at wound time = at48 time h. Scale = 48 barh. Scale= 500 barµm. = 500 μm.

FigureFigure 7. Images 7. Images of theof the wounds wounds at diat ﬀdifferenterent time time points points after after removing removing thethe barrier.barrier. ( (aa)) PBS PBS-coated-coated and uncoatedand uncoated surfaces. surfaces. (b) Poly- (bl)-lysine-coated Poly-L-lysine-coated and uncoated and uncoated surfaces. surfaces. (c) Gelatin-coated (c) Gelatin-coated and uncoated and uncoated surfaces. Scale bar = 500 μm. µ Figuresurfaces. 7. ScaleImages bar of= the500 woundsm. at different time points after removing the barrier. (a) PBS-coated and uncoated surfaces. (b) Poly-L-lysine-coated and uncoated surfaces. (c) Gelatin-coated and uncoated surfaces. Scale bar = 500 μm.

Materials 2019, 12, 2775 8 of 10 Materials 2019, 12, x FOR PEER REVIEW 8 of 10

Figure 8. 8. (a) (Calculateda) Calculated wound wound-healing-healing rates ratesunder underall conditions. all conditions. Statistical Statisticalanalysis was analysis performed was onperformed nine independent on nine independent data points data (see Sectionpoints (see 2.5).Section Student’s 2.5 ). t-tests Student’s were performedt-tests were between performed (b) gelatinbetween-coated (b) gelatin-coated and uncoated and surfaces, uncoated (c) surfaces, poly-L-lysine (c) poly--coatedl-lysine-coated and uncoated and uncoated surfaces, surfaces, and (d) PBSand- (coatedd) PBS-coated and uncoated and uncoated surfaces surfaces.. ns: no ns:statistically no statistically significant signiﬁcant difference diﬀerence (p > 0.05) (p > 0.05);; *: p *:< p0.05;< 0.05; **: p**:

4. Conc Conclusionslusions InIn this study, we reported a modifiedmodified barrier assay for studying the effects effects of substrate-coatingsubstrate-coating materials on the wound wound-healing-healing process. The The migration migration of of fibroblasts fibroblasts on on both coated and uncoated surfaces was was followed followed simultaneously simultaneously in one in single one petri single dish. petri This dish. greatly This eliminated greatly the eliminated experimental the experimentalrun-to-run variations. run-to-run The variations. results indicated The results that (1) indicated both poly- thatl-lysine (1) both and gelatin poly-L coatings-lysine and had gelatin almost coatingsno effects had on cellalmost proliferation no effects and on viability,cell proliferation and (2) compared and viability, to the and uncoated (2) compared one, cells to migrated the uncoated faster one,on coated cells migrated surfaces byfaster showing on coated increased surfaces wound-healing by showing increased rates. We wound will be- workinghealing rates. on investigating We will be workingthe effects on of investigating other coating the materials effects of suchother ascoating collagen, materials fibronectin, such as laminin,collagen, and fibronectin, poly-l-ornithine. laminin, andThe presentpoly-L- platformornithine. is The of help present in further platform understanding is of help thein mechanismsfurther underst of substrateanding the coating-dependent mechanisms of substratewound-healing coating processes.-dependent wound-healing processes.

Author Contributions: Conceptualization, K.-Y.L. and Y.-S.S.; methodology, J.-Y.L. and Y.-S.S.; validation, J.-Y.L; Author Contributions: Conceptualization, K.-Y.L. and Y.-S.S.; methodology, J.-Y.L. and Y.-S.S.; validation, investigation, J.-Y.L.; data curation, J.-Y.L.; writing—original draft preparation, Y.-S.S.; writing—review and J.editing,-Y.L; investigation, Y.-S.S. J.-Y.L.; data curation, J.-Y.L.; writing—original draft preparation, Y.-S.S.; writing—review and editing, Y.-S.S. Funding: This research was funded by the Ministry of Science and Technology, Taiwan, grant number MOST Funding:106-2112-M-030-001-MY2 This research was and funded MOST by 107-2221-E-030-001-MY2. the Ministry of Science and Technology, Taiwan, grant number MOST 106Conﬂicts-2112-M of-030 Interest:-001-MY2The and authors MOST declare 107-2221 no conﬂict-E-030- of001 interest.-MY2. Conflicts of Interest: The authors declare no conflict of interest. References

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