research article the favorable effect of...

Research ArticleThe Favorable Effect of Mesenchymal Stem CellTreatment on the Antioxidant Protective Mechanism inthe Corneal Epithelium and Renewal of Corneal OpticalProperties Changed after Alkali Burns

Cestmir Cejka1 Vladimir Holan23 Peter Trosan23 Alena Zajicova2

Eliska Javorkova23 and Jitka Cejkova1

1Laboratory of Eye Histochemistry and Pharmacology Institute of Experimental Medicine Academy of Sciences of the Czech RepublicVidenska 1083 14220 Prague 4 Czech Republic2Department of Transplantation Immunology Institute of Experimental Medicine Academy of Sciences of the Czech RepublicVidenska 1083 14220 Prague 4 Czech Republic3Faculty of Natural Science Charles University Vinicna 7 12843 Prague 2 Czech Republic

Correspondence should be addressed to Jitka Cejkova cejkovabiomedcascz

Received 16 October 2015 Accepted 16 November 2015

Academic Editor Janusz Gebicki

Copyright copy 2016 Cestmir Cejka et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The aim of this study was to examine whether mesenchymal stem cells (MSCs) andor corneal limbal epithelial stem cells (LSCs)influence restoration of an antioxidant protective mechanism in the corneal epithelium and renewal of corneal optical propertieschanged after alkali burns The injured rabbit corneas (with 025N NaOH) were untreated or treated with nanofiber scaffolds freeof stem cells with nanofiber scaffolds seeded with bone marrowMSCs (BM-MSCs) with adipose tissueMSCs (Ad-MSCs) or withLSCs On day 15 following the injury after BM-MSCs or LSCs nanofiber treatment (less after Ad-MSCs treatment) the expression ofantioxidant enzymeswas restored in the regenerated corneal epitheliumand the expressions ofmatrixmetalloproteinase 9 (MMP9)inducible nitric oxide synthase (iNOS) 120572-smooth muscle actin (120572-SMA) transforming growth factor-1205731 (TGF-1205731) and vascularendothelial factor (VEGF) were low The central corneal thickness (taken as an index of corneal hydration) increased after theinjury and returned to levels before the injury In injured untreated corneas the epithelium was absent and numerous cells revealedthe expressions of iNOS MMP9 120572-SMA TGF-1205731 and VEGF In conclusion stem cell treatment accelerated regeneration of thecorneal epithelium restored the antioxidant protective mechanism and renewed corneal optical properties

1 Introduction

Alkali injury of the cornea threatens vision Immediatelyafter the alkali injury oxidative stress appears in the corneawhich precedes the corneal inflammatory response [1 2]Following alkali injury of the cornea or after irradiation of thecornea withUVB rays the antioxidantprooxidant imbalanceappeared in the damaged corneal epitheliumThe expressionand activities of corneal antioxidant enzymes decreasedwhereas the expression and activities of prooxidant enzymesremained at physiological levels or even increased [3 4]Reactive oxygen species (ROS) were insufficiently cleaved

According to Finkel and Holbrook [5] ROS can activatethe transcription factor NF-kB which then translocate tothe nucleus to induce the production of proinflammatorycytokines highly involved in the development of inflamma-tion ROS contribute to the induction of various enzymessuch as metalloproteinases serine proteases and nitric oxidesynthases [6 7] Toxic oxygen and nitrogen products withproteolytic enzymes degrade the cornea [8 9]The immediatestart of topical treatment of the alkali-injured eye withantioxidant H2-enriched irrigation solution and N-acetyl-L-cysteine [1] or with bone marrow mesenchymal stem cells(BM-MSCs) revealing antioxidant properties significantly

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2016 Article ID 5843809 12 pageshttpdxdoiorg10115520165843809

2 Oxidative Medicine and Cellular Longevity

suppressed intracorneal inflammation and corneal neovascu-larization [2] It is well known that MSCs possess antioxidantproperties [2 10ndash13] The beneficial antioxidant effects ofMSCs may be explained by the findings that MSCs secretesuperoxide dismutase [14] Superoxide dismutase belongsto antioxidant enzymes that dismute toxic free radicalsproduced during inflammation [15]

To our knowledge this is the first paper investigatingthe effects of bone marrow MSCs (BM-MSCs) adiposetissue MSCs (Ad-MSCs) or corneal limbal epithelial stemcells (LSCs) on the antioxidant protective mechanism inthe regenerated corneal epithelium and on corneal opticalproperties evaluated by central corneal thickness (taken asan index of corneal hydration) and corneal transparency Wefound that in contrast to the effect of nanofibers alone (MSCs-free) where profound enzymatic antioxidantprooxidantimbalance was found in the corneal epithelium after thetreatment of injured corneas with BM-MSCs nanofibersor LSCs nanofibers (less after Ad-MSCs nanofibers) theenzymatic antioxidant protective mechanism was restored inthe regenerated corneal epithelium This was accompaniedby the renewal of corneal optical properties and acceleratedcorneal healing

2 Material and Methods

21 Preparation of StemCells LSCs were obtained by enzymedigestion of rabbit limbal tissue as we have described ina mouse model [16] In brief limbal tissue was cut withscissors and subjected to 10 short (10min each) trypsinizationcycles The released cells were harvested after each cyclecentrifuged (8 minutes at 250 g) and resuspended in RPMI1640 medium (Sigma St Louis MO) containing 10 fetalcalf serum (FCS Sigma) antibiotics (100UmL of penicillin100 120583gmL of streptomycin) and 10mM HEPES buffer Thecells were seeded in 25 cm2 tissue culture flasks (CorningSchiphol-Rijk Netherlands) For characterization of cells andfor their transfer to the nanofiber scaffold the cells growingin vitro for 2-3 weeks (3rd passage) were used

BM-MSCs were isolated from femurs of rabbitsThe bonemarrowwas flushed out a single cell suspensionwas preparedby homogenization and the cells were seeded at a concen-tration of 4 times 106 cellsmL in Dulbeccorsquos modified Eaglersquosmedium (DMEM Sigma) containing 10 FCS antibiotics(100UmL of penicillin 100mgmL of streptomycin) and10mMHEPES in 25 cm2 tissue culture flasks (Corning) Aftera 48-hour incubation the nonadherent cells were washed outand the adherent cells were cultured with a regular exchangeof the medium and passaging of the cells to maintain theiroptimal concentrationThe cells were characterized and usedafter the 3rd passage

Ad-MSCs were isolated from subcutaneous adipose tis-sue The tissue was cut into small pieces with scissors andincubated in 1mL of Hankrsquos balanced salt solution containing10mgmLof collagenase type I (Sigma) for 60minutes at 37∘Cwith gentle agitation Then the collagenase was diluted withcomplete DMEM The cells were filtered and centrifuged at250 g for 8 minutes The upper adipose layer was removedand cells were centrifuged resuspended in 6mL of complete

DMEM(4times 106 cellsmL) and seeded in 25 cm2 tissue cultureflasks (Corning) After incubation for 48 hours the cells werewashed with a medium to remove nonadherent cells and celldebris and cultured under standard conditions Ad-MSCswere used in passages 3-4

22 StemCell Differentiation andGene Expression Theabilityof stem cells to differentiate into other cell types was deter-mined according to the ability to differentiate into adipocytesusing specific adipogenic medium containing 01 120583M dex-amethasone 05mM 3-isobutyl-1-methylxanthine 01mMindomethacin and 05 120583gmL of insulin as we describedpreviously [17] Differentiation of cells was confirmed bystaining with Oil Red O

The expression of genes for the immunoregulatorymolecules indoleamine-23-dioxygenase (IDO) cyclooxy-genase-2 (Cox-2) and transforming growth factor-120573 (TGF-120573) was determined in unstimulated and lipopolysaccharide-(LPS-) stimulated MSCs and LSCs In these experiments thecells (4 times 104 cellswell) were cultured in 700120583L of DMEMfor 48 hours in 24-well tissue culture plates (Corning) with orwithout 5 120583gmL of LPS and the expression of the genes wasdetermined by real-timePCR aswe have described elsewhere[18]

23 Nanofiber Scaffold Preparation The PLA polymer waspurchased from Nature Works LLC (Minnetonka Min-neapolis Minnesota USA) This material was dissolved inchloroform at 7weight percent (wt) and two other solvents12-dichloroethane (29wt) and ethyl acetate (10 wt) (bothpurchased from PENTA Prague Czech Republic) wereadded to this solution The mixture was stirred until ahomogenous polymer solution was obtained The modifiedneedleless Nanospider technology in which polymeric jetsare spontaneously formed from liquid surfaces on a rotatingspinning electrode was used to prepare the nanofibersNanofiber scaffolds were cut into squares (approximately 15times 15 cm) and fixed into CellCrown24 inserts (Scaffdex) Theinserts with nanofibers were transferred into 24-well tissueculture plates (Corning) Three times 105 stem cells in a volumeof 700120583L of complete DMEMwere transferred into eachwellThe plates were incubated for 24 hours to allow cells to adhereto the scaffold [19]

24 Alkali Injury of the Cornea in Experimental Animals andNano Scaffold Transfer Adult female New Zealand whiterabbits (25ndash30 kg) were used in our experiments The inves-tigation was conducted according to the ARVO Statementon the Use of Animals in Ophthalmic and Vision ResearchRabbits were anesthetized by an intramuscular injection ofRometar (xylazinum hydrochloricum Spofa Prague CR2 02mLkg body weight) and Narkamon (ketaminumhydrochloricum Spofa 5 1mLkg body weight)

Sodium hydroxide (025N NaOH) was applied by meansof dropping on the corneal surface (10 drops during 1min)then the eyes were immediately rinsed with tap water Theanimals were divided into five groups (each group containedsix rabbits) In the first group of animals the injured eyeswere

Oxidative Medicine and Cellular Longevity 3

left without further treatment In the second group drug-free nanofiber scaffoldswere transferred onto the injured eyesand sutured to the conjunctiva and the eyelids closed In thethird group BM-MSCs nanofibers were transferred onto theinjured corneal surface and sutured to the conjunctiva andthe eyelids closed In the fourth group Ad-MSCs nanofiberswere transferred with cell side facing down on the alkali-injured corneal surface and sutured to the conjunctiva andthe eyelids closed In the fifth group LSCs nanofibers weretransferred to the corneal surface and sutured to the conjunc-tiva and the eyelids closed

The scaffolds were transferred onto the ocular sur-face immediately after the injury and sutured to the con-junctiva with four interrupted sutures using 110 Ethilon(Ethicon Johnson amp Johnson Livingston England) Theeyelids were closed by tarsorrhaphy using 1 suture of Resolon70 (Resorba Nuremberg Germany) for 72 hours Anophthalmic ointment compound containing bacitracin andneomycin (Ophthalmo-Framykoin Zentiva Prague CzechRepublic) was appliedThe nanofiber scaffolds were removedfrom the ocular surface on day 4 after the operation After thealkali injury and awakening from the anesthesia the rabbitswere treated with analgesia (ketoprofen 10mgkg im) twotimes daily for five days The animals were sacrificed fol-lowing an iv injection of thiopental anesthesia (ThiopentalSpofa 30mgkg) after premedication with an intramuscularinjection of RometarNarkamon on day 15 after the injuryIn all experiments with alkali injury the corneas of healthyrabbit eyes served as controls Photographs of the corneaswere taken throughout the whole experiment

25 Microscopical Examinations After sacrificing the ani-mals the eyes were enucleated and the anterior eye segmentsdissected out and quenched in light petroleum chilledwith anacetone-dry ice mixture Sections were cut on a cryostat andtransferred to glass slides Subsequently the cryostat sectionswere fixed in acetone at 4∘C for 5 minutes For the immuno-histochemical localization of xanthine oxidase (XOX) super-oxide dismutase (SOD) glutathione peroxidase (GPX) cata-lase (CAT) 120572-smooth muscle actin (120572-SMA) induciblenitric oxide synthase (iNOS) matrix metalloproteinase-9 (MMP9) interleukin-6 (IL-6) and vascular endothelialgrowth factor (VEGF) the following primary antibodieswere used mouse monoclonal anti-xanthine oxidase Ab-2 (NeoMarkers Fremont CA USA) mouse monoclonalanti-superoxide dismutase sheep polyclonal anti-catalasesheep polyclonal anti-glutathione peroxidase (BiogenesisPoole UK) goat polyclonal anti-matrix metalloproteinase-9 (Santa Cruz Biotechnology Santa Cruz CA USA) mousemonoclonal anti-120572-SMA (Sigma Saint Louis MO USA)mouse monoclonal anti-human iNOS (Biosciences San JoseCA USA) goat polyclonal anti-MMP9 (Santa Cruz Biotech-nology Santa Cruz CA USA) mouse monoclonal anti-IL-6 (Abcam Cambridge UK) and mouse monoclonal anti-VEGF (Abcam Cambridge UK)The binding of the primaryantibodies was demonstrated using the HRPDAB UltraVision detection system (Thermo Scientific Fremont CA)following the instructions of the manufacturer hydrogenperoxide block (15 minutes) ultra V block (5 minutes)

primary antibody incubation (60 minutes) biotinylated goatanti-mouse IgG (Lab Vision Fremont CA) or donkey anti-goat IgG (Santa Cruz Biotechnology) secondary antibodyincubation (10min) and peroxidase-labeled streptavidinincubation (10 minutes) Visualization was performed usinga freshly preparedDAB substrate-chromogen solution Cryo-stat sections in which the primary antibodies were omittedfrom the incubation media served as negative controls Somesections were counterstained with Mayerrsquos hematoxylin

26 Determination of Corneal Thickness Changes of cornealoptical properties after the injury and during healing wereevaluated by measuring the central corneal thickness (takenas an index of corneal hydration) [20] The central cornealthickness was measured in anesthetized animals using anultrasonic pachymeter SP-100 (Tomey Corporation NagoyaJapan) in the corneal center The corneal thickness wasmeasured in the same corneas before alkali injury (corneasof healthy eyes) and five and fifteen days after the injury (allexperimental groups) Each cornea was measured four timesand the mean value of the thickness (in 120583m) was computed

27 Evaluation of Corneal Neovascularization and Trans-parency For evaluation of corneal neovascularization thenumber of vessels was counted in each of 60∘ sectors of thecorneal surfaceThemean value and standard deviation werecounted from fivemeasurementsThis procedure was appliedfor every eye from a matching group of eyes (control injureduntreated injured and treated with drug-free nanofibers andinjured and treated with MSCs nanofibers or with Ad-MSCsnanofibers or with LSCs nanofibers)

28 Detection of Gene Expression by Real-Time PCR Theexpression of genes for TGF-1205731 VEGF and iNOS in con-trol and treated corneas was determined by quantitativereal-time polymerase chain reaction (PCR) Corneas wereexcised using Vannas scissors transferred into Eppendorftubes and immediately frozen The frozen corneal tissuewas then homogenized and added in 500120583L of TRI Reagent(Molecular Research Center Cincinnati OH) for the RNAisolationThe details of RNA isolation transcription and thePCR parameters have been described previously [21] In brieftotal RNA was extracted using TRI Reagent according to themanufacturerrsquos instructions One 120583g of total RNAwas treatedusing deoxyribonuclease I (Promega) and subsequently usedfor reverse transcription The first-strand cDNA was syn-thesized using random primers (Promega Madison WI)in a total reaction volume of 25 120583L using M-MLV ReverseTranscriptase (Promega)

Quantitative real-time PCRwas performed in a StepOne-Plus real-time PCR system (Applied Biosystems)The relativequantification model with efficiency correction was appliedto calculate the expression of the target gene in comparisonwith GAPDH used as the housekeeping gene The followingprimers were used for amplification GAPDH 51015840-CCCAAC-GTGTCTGTCGTG (sense) 51015840-CCGACCCAGACGTAC-AGC (antisense) iNOS 51015840-AGGGAGTGTTGTTCCAGG-TG (sense) 51015840-TCCTCAACCTGCTCCTCACT (antisense)


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Figure 1The ability of cells to differentiate into adipocytes was characterizedmicroscopically ((a) upper undifferentiated cells lower cells indifferentiationmedium) Expression of genes for immunoregulatorymolecules by BM-MSCs Ad-MSCs and LSCs (b)The cells were culturedfor 48 hours unstimulated or stimulated with LPS and the expression of genes for IDO TGF-120573 and Cox-2 was determined by real-time PCREach bar represents the mean plusmn SD from 5 determinations

TGF-1205731 51015840-GCCTGCAAGTGCTCAAGTTAC (sense) 51015840-TGCTGCATTTCTGGTACAGC (antisense) and VEGF51015840-CGAGACCTTGGTGGACATCT (sense) 51015840-ATCTGC-ATGGTGACGTTGAA (antisense) The PCR parametersincluded denaturation at 95∘C for 3min then 40 cycles at95∘C for 20 s annealing at 60∘C for 30 s and elongation at72∘C for 30 s Fluorescence data were collected at each cycleafter an elongation step at 80∘C for 5 s and were analyzedon the StepOne Software version 222 (Applied Biosystems)Each individual experiment was done in triplicate

29 Statistics An analysis of the data showed normal dis-tribution and the results are expressed as mean plusmn SDComparisons between the two groupsweremade by Studentrsquost-test and multiple comparisons were analyzed by ANOVAA value of 119875 lt 005 was considered statistically significant

3 Results

31 Differentiation and Gene Expression of Rabbit MSCs andLSCs When all three cell types were cultured in a specific

adipogenic differentiation medium the highest differentia-tion potential was recorded in BM-MSCs as demonstratedmicroscopically (Figure 1(a)) To test the ability of BM-MSCs Ad-MSCs and LSCs to express genes for the basicimmunoregulatory molecules the cells were cultured for 48hours unstimulated or stimulated with LPS and the expres-sion of genes for IDO-2 iNOS and Cox-2 was determinedby real-time PCR As demonstrated in Figure 1(b) the genefor TGF-1205731 was expressed spontaneously and comparablyin all cell types while the genes for IDO and Cox-2 wereexpressed preferentially in only some cell populations or onlyafter stimulation with LPS

32 Immunohistochemical Detection of SOD XOX MMP9and iNOS and Gene Expression of iNOS in the Alkali-Injuredand Stem Cell Treated Corneas (Day 15 after the Injury)The expressions of SOD and XOX were balanced in injuredcorneas treated with BM-MSCs nanofibers (Figures 2(c) and2(d)) and LSCs nanofibers (Figures 2(g) and 2(h)) similarto the control (healthy corneas) (Figures 2(i) and 2(j))In injured corneas treated with Ad-MSCs the expression


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Figure 2 The immunohistochemical detection of SOD and XOX in the corneal epithelium in injured and stem cell treated corneas on day15 after the injury In BM-MSCs treated corneas (c d) and LSCs treated corneas (g h) expressions of SOD and XOX are relatively high andbalanced similar to the control corneas (i j) In contrast in Ad-MSCs treated corneas (e f) and Nano-treated corneas (a b) the expressionsof SOD are decreased compared to controls (i) Scale bars 50 120583m

of SOD (Figure 2(e)) was decreased compared to controls(Figure 2(i)) The expression of SOD was also decreased(compared to controls) in injured corneas treated with cell-free nanofibers (Figure 2(a)) Similar results (as with SOD)were obtained with the expression of CAT and GPX (images

not shown) Also in these cases the balance between theexpressions of CAT and XOX and GPX and XOX wasobtained only in injured corneas treated with BM-MSCsnanofibers or LSCs nanofibers The expressions of CATand GPX were decreased (compared to controls) after the


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nFigure 3The expression of MMP9 and iNOS in injured and stem cell treated corneas on day 15 after the injuryThe expression of MMP9 (a)and iNOS (b) in untreated corneas without epithelium and in Nano-treated reepithelialized corneas (c d) is high and decreases in Ad-MSCstreated corneas (g h) In corneas treated with BM-MSCs (e f) or LSCs (i j) the expressions of MMP9 are low and the expression of iNOS isnearly absent similar to the control cornea (k l)The corneal sections are counterstained with hematoxylin Scale bars 50 120583mThe expressionof genes for iNOS (m) on day 15 after the injury was determined by real-time PCR Each bar represents the mean plusmn SD from 6 individualcorneas The values with asterisks represent statistically significant (lowast119875 lt 005) difference from untreated injured corneas

treatment of injured corneas with Ad-MSCs or Nano freeof MSCs The expression of MMP9 (Figure 3(a)) or iNOS(Figure 3(b)) was high in injured untreated corneas andonly less decreased in injured corneas treated with cell-freenanofibers (Figures 3(c) and 3(d)) However the treatment ofinjured corneas with BM-MSCs nanofibers (Figures 3(e) and3(f)) or with LSCs nanofibers (Figures 3(i) and 3(j)) (less afterAd-MSCs nanofibers Figures 3(g) and 3(h)) suppressed theexpression of MMP9 and iNOS in corneas The expressionof MMP9 (Figure 3(k)) was very low in control corneasand the expression of iNOS (Figure 3(l)) was absent Theexpression of genes for iNOS in healthy injured untreatedand injured treated corneas was quantified by real-time PCR(Figure 3(m)) In accordance with the results from immuno-histochemistry treatment of injured corneas with BM-MSCnanofibers or LSC-seeded nanofiber scaffolds decreased theiNOS expressions

33 Immunohistochemical Detection of 120572-SMA and VEGFand Gene Expression of TGF-120573 in Alkali-Injured and StemCell Treated Corneas (Day 15 after the Injury) In injureduntreated corneas (Figure 4(a)) and corneas treated withstem-cell-free nanofibers (Figure 4(b)) the expression of 120572-SMA was high whereas in injured corneas treated with BM-MSCs nanofibers (Figure 4(c)) with LSCs (Figure 4(e)) orwithAd-MSCs nanofibers (Figure 4(d)) the expressionswerelow In control (healthy) corneas (Figure 4(f)) the expressionof 120572-SMA was absent The expression of genes for TGF-1205731in healthy and injured untreated and treated corneas withstem cells was quantified by real-time PCR (Figure 4(g))Thetreatment of injured corneas with stem cells decreased theexpression of TGF-1205731 The expression of VEGF was very lowin healthy control corneas (Figure 5(f)) On day 15 after theinjury the expression of VEGFwas high in untreated corneas(Figure 5(a)) and was only less decreased in corneas treatedwith cell-free nanofiber scaffold (Figure 5(b)) The treatmentof injured corneas with Ad-MSC-seeded nanofibers reduced

VEGF expression (Figure 5(d)) The highest reduction ofVEGF expression was seen in corneas treated with BM-MSCs nanofiber scaffolds (Figure 5(c)) or LSCs nanofibers(Figure 5(e)) The expression of genes for VEGF in control(healthy) injured untreated and injured treated corneas wasquantified by real-time PCR (Figure 5(g)) The treatment ofinjured corneas with stem-cell-seeded nanofiber scaffoldssignificantly decreased the expression of VEGF

34 Corneal Opacity of Alkali-Injured and Stem Cell TreatedEyes Representative photographs of healthy injured andtreated eyes are shown in Figure 6 In comparison with thehealthy control eyes (Figure 6(a)) corneas of injured eyesbecame opalescent following the injury (Figure 6(b)) andremained opalescent and highly vascularized on day 15 afterthe injury (Figure 6(d)) The eye covered with nanofiberscaffold immediately after the injury is shown in Figure 6(c)Some improvement of corneas was observed on day 15 afterthe injury in eyes treated with cell-free nanofiber scaffold(Figure 6(e)) however corneas were vascularized In the eyestreated with stem-cell-seeded nanofibers the corneal opacitywas decreased and corneal neovascularizationwas apparentlyless expressed (Figures 6(f)ndash6(h)) with the best therapeuticeffects of the treatment with BM-MSCs (Figure 6(f)) or withLSCs (Figure 6(h)) Quantification of corneal neovascular-ization is summarized in Figure 6(i) The number of vesselswas high in injured untreated corneas and was partiallyreduced in injured corneas treated with cell-free nanofibersTreatment of injured corneas with nanofiber scaffolds seededwith all three types of stem cells significantly decreasedcorneal neovascularization The highest decrease was foundin injured corneas treated with nanofiber scaffolds seededwith BM-MSCs or with LSCs

35 Central Corneal Thickness after Alkali Injury and Treat-ment with Stem Cells (Figure 7) Shortly after alkali injurythe central corneal thickness increased more than twice


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Figure 4The expression of 120572-SMA and TGF-1205731 in injured and stem cell treated corneas on day 15 after the injury The expression of 120572-SMAis high in untreated (a) and Nano-treated (b) corneas whereas in corneas treated with BM-MSCs (c) Ad-MSCs (d) and LSCs (e) it is lowCompare with control cornea (f) where the expression of 120572-SMA is absent The corneal sections are counterstained with hematoxylin Scalebars 50 120583m The expression of genes for TGF-1205731 (g) on day 15 after the injury was determined by real-time PCR Each bar represents themean plusmn SD from 6 individual corneas The values with asterisks represent a statistically significant (lowast119875 lt 005 lowastlowast119875 lt 001) difference fromuntreated injured corneas

(compared to levels before injury day 0) and remained highon day 5 in untreated corneas or in corneas treated withcell-free nanofiber scaffoldThe central corneal thickness wassignificantly reduced already on day 5 in corneas treated withnanofiber scaffolds seeded with stem cells On day 15 afterinjury the corneal thickness remained increased in untreatedinjured corneas but was profoundly decreased in corneastreated with stem-cell-seeded nanofibers On day 15 after theinjury in corneas treatedwith nanofiber scaffolds seededwith

BM-MSCs or LSCs the corneal thickness returned to thevalues before injury (day 0)

4 Discussion

In this paper the stem cells were incubated and transferredonto the damaged corneal surface withCsA-loaded nanofiberscaffoldsThe nanofiber scaffolds are biocompatible and have


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Figure 5 The expression of VEGF in injured and stem cell treated corneas on day 15 after the injury VEGF expression is high in untreatedcorneas (a) and Nano-treated corneas (b) and less expressed in Ad-MSCs treated corneas (d) After the treatment of corneas with BM-MSCs(c) and LSCs (e) the expression of VEGF is low In control corneas (f) the expression of VEGF is absentThe sections are counterstained withhematoxylin Scale bars 50 120583mThe expression of genes for VEGF (g) on day 15 after the injury was determined by real-time PCR Each barrepresents the mean plusmn SD from 6 individual corneas The values with asterisks represent a statistically significant (lowast119875 lt 005) difference fromuntreated injured corneas

good mechanical properties to fixate on the ocular surfaceThe stem cells migrate from the scaffolds onto the ocularsurface and support corneal healing [22] Moreover CsA-loaded nanofibers are synthetic and for this reason moresuitable in many cases than biomaterials such as collagensheets or amniotic membrane These biomaterials have somelimitations such as enzymatic digestion of collagen sheets[23] or the presence of anti- as well as proinflammatorycytokines (IL-6 IL-8) or metalloproteinases in amniotic

membranes which may in some cases controversially con-tribute to cornealmelting [24] Using CsA-loaded nanofibersour results show that the treatment of alkali-injured corneaswith BM-MSCs nanofibers or LSCs nanofibers (less with Ad-MSCs nanofibers) accelerated corneal reepithelialization andrestored the antioxidantprooxidant balance in the regener-ated corneal epithelium This was accompanied by cornealhealing with renewed optical properties and highly reducedneovascularization


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Figure 6 Corneal opacity of alkali-injured and stem cell treated eyes Representative photographs show a control healthy eye (a) alkali-injured eye (immediately after the injury) (b) the injured eye with sutured nanofiber scaffold (immediately after the injury) (c) and injuredeyes on day 15 after the injury which were untreated (d) or treated with cell-free nanofiber scaffold (e) or with nanofiber scaffolds seededwith BM-MSCs (f) Ad-MSCs (g) or LSCs (h) Corneal neovascularization is strongly expressed in untreated corneas ((d) arrows) and highlysuppressed in corneas treated with nanofiber scaffolds BM-MSCs (f) and LSCs (h)The quantification of corneal neovascularization is shownin (i) Each bar represents the mean plusmn SD from 6 corneas The values with asterisks are significantly different (lowastlowastlowast119875 lt 0001) from untreatedinjured corneas

The antioxidantmechanism located in the corneal epithe-lium is very important for the protection of the corneaagainst toxic environmental influences After the injury withalkali or irradiation of the cornea with UVB rays thelevels of corneal antioxidant enzymes (SOD CAT and GPX)decreased in the corneal epithelium whereas the levels ofprooxidant enzymes oxidases that generate ROS with themost important role of XOX remained at physiological levelsor even increased [2ndash4] ROS generated in high numberswere insufficiently cleaved This was dangerous to the corneabecause ROS induce proinflammatory cytokine generationleading to the increased expression and activation of destruc-tive enzymes damaging the cornea [25] According to theconcentration of alkali and extent of injury corneas ulcerate

or perforate during 2 to 3 weeks after the injury or corneasslowly heal with untransparent scar formation [26] From ourprevious studies (unpublished results) it followed that afterthe injury of rabbit corneas with 025N NaOH (by means ofdropping on the corneal surface) the corneal epithelium waslost after the injury and during the subsequent 15 days theepithelium was not regenerated in untreated injured corneasTherefore this time interval was chosen in this study (as theend of experiment) using a similar concentration of alkali andmode of application We found that on day 15 the after theinjury the untreated corneas were not reepithelialized Thiswas in contrast to injured corneas treated with nanofibersalone (MSCs-free) which were reepithelialized however theexpressions of antioxidant enzymes were decreased in the


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800

1000

UntreatedNanoNano BM-MSCs

Nano LSCsNano Ad-MSCs


lowastlowastlowast lowast

lowastlowastlowastlowastlowastlowastlowastlowastlowast

ns lowastlowastlowastlowastlowastlowast

ns lowastlowastlowastns

Days after injury

Cen

tral

corn

eal t

hick

ness

(120583m

)

Figure 7 Central corneal thickness of healthy alkali-injured andstem cell treated corneas The corneas were injured with alkaliand untreated or treated with nanofiber scaffold alone (Nano)or with nanofiber scaffolds seeded with BM-MSCs Ad-MSCs orLSCs Central corneal thickness was measured in the same rabbitbefore injury (day 0) and on days 5 and 15 after the injury Eachbar represents the mean plusmn SD from 6 corneas The values (blackasterisks) for day 5 are statistically different from untreated injuredcorneas on day 5 similarly the values for day 15 (black asterisks)are statistically different from untreated injured corneas on day 15(lowast119875 lt 005 lowastlowastlowast119875 lt 0001) (ns not significant)The values for day 15(red asterisks) are statistically different from the values before injuryday 0 (lowastlowastlowast119875 lt 0001) (ns not significant)

regenerated corneal epithelium (compared to healthy controlcorneas) In contrast after BM-MSCs nanofiber or LSCsnanofiber treatment the expressions of antioxidant enzymeswere restored and balanced with prooxidant enzymes inthe epithelium similar to the control (healthy) corneas(Figure 2)

In this study BM-MSCs Ad-MSCs and LSCs were pre-pared from the same rabbit and their growth differentiationproperties and the ability to produce immunoregulatoryand growth factors were compared (Figure 1) Besides theimmunomodulatory and anti-inflammatory properties ofMSCs these stem cells reveal important antioxidant effectsMSCs can exert direct antioxidant activities through thesecretion of antioxidant moleculesThey secrete the extracel-lular antioxidant molecule superoxide dismutase 3 (SOD3)[14 27] The superoxide dismutase family member SOD3 isthe only antioxidant enzyme that scavenges superoxide inthe extracellular space and reduces the formation of toxicoxygen and nitrogen products such as the hydroxyl radicaland peroxynitrite In our experiments stem cells on nanofiberscaffolds were transferred immediately after the injury tothe damaged ocular surface It is suggested that the SOD3of stem cells cleaved ROS generated for example by XOXbecause both SOD3 and ROS were released extracellularly[4] This may be one mechanism decreasing the amount ofROS and supporting corneal reepithelialization and healingIn injured corneas treatedwith stem cells seeded on nanofiberscaffolds the expression and levels of MMP9 iNOS 120572-SMATGF-1205731 and VEGF were significantly decreased comparedto untreated injured corneas or injured corneas treated with

Nano free of MSCs (Figures 3ndash5) It has been demonstratedin a variety of experimental studies that SOD3 can attenuatetissue damage and inflammation [28 29] The ability oftransplanted LSCs to regenerate corneal epithelium [30] iswell known This is also shown in our study However wefirst demonstrate in this study the renewal of the expressionof antioxidant enzymes in the regenerated corneal epitheliumafter the treatment of alkali-injured corneas with stem cells

5 Conclusions

BM-MSCs nanofibers and LSCs nanofibers (less Ad-MSCsnanofibers) transferred onto the damaged corneal surfaceimmediately after the injury accelerated corneal reepithelial-ization and restored the antioxidantprooxidant balance inthe regenerated corneal epithelium Corneal inflammationwas suppressed and corneal neovascularization reduced Thecentral corneal thickness (taken as an index of corneal hydra-tion) reached levels before injury Corneal optical propertieswere renewed to a large extent Although there are somedifferences between the human and rabbit cornea in therecovering of endothelial function (faster in rabbits thanksto cell proliferation) the efficacy of stem cell treatment ofalkali-injured cornea on the renewal of physiological levelsof corneal hydration and on the restoration of transparencycompared to untreated injured corneas or injured corneastreated with nanofibers free of cells was clearly shown in ourrabbit model

Abbreviations

BM-MSCs Bone marrow mesenchymal stem cellsAd-MSCs Adipose tissue mesenchymal stem cellsLSCs Limbal corneal epithelial stem cellsROS Reactive oxygen speciesiNOS Inducible nitric oxide synthaseMMP9 Matrix metalloproteinase-9120572-SMA Alpha-smooth muscle actinTGF-1205731 Transforming growth factor-beta 1VEGF Vascular endothelial growth factor

Conflict of Interests

The authors declare that there is no conflict of interestsregarding publication of this paper

Acknowledgments

This work was supported by Grants 14-12580S from the GrantAgency of the Czech Republic and 889113 from the GrantAgency of Charles University and Projects CZ1051100020109 CZ216310021528 NPUI LO1309 and LO1508UNCE 204013 and SVV 260206

References

[1] M Kubota S Shimmura S Kubota et al ldquoHydrogen and N-acetyl-L-cysteine rescue oxidative stress-induced angiogenesis


in a mouse corneal alkali-burn modelrdquo Investigative Ophthal-mology and Visual Science vol 52 no 1 pp 427ndash433 2011

[2] J Cejkova P Trosan C Cejka et al ldquoSuppression of alkali-induced oxidative injury in the cornea by mesenchymal stemcells growing on nanofiber scaffolds and transferred onto thedamaged corneal surfacerdquo Experimental Eye Research vol 116no 5 pp 312ndash323 2013

[3] J Cejkova S Stipek J Crkovska and T Ardan ldquoChangesof superoxide dismutase catalase and glutathione peroxidasein the corneal epithelium after UVB rays Histochemical andbiochemical studyrdquo Histology and Histopathology vol 15 no 4pp 1043ndash1050 2000

[4] J Cejkova S Stıpek J Crkovska T Ardan and A MidelfartldquoReactive oxygen species (ROS)-generating oxidases in thenormal rabbit cornea and their involvement in the cornealdamage evoked byUVB raysrdquoHistology andHistopathology vol16 no 2 pp 523ndash533 2001

[5] T Finkel and N J Holbrook ldquoOxidants oxidative stress and thebiology of ageingrdquoNature vol 408 no 6809 pp 239ndash247 2000

[6] J Cejkova T Ardan C Cejka J Kovaceva and Z ZıdekldquoIrradiation of the rabbit cornea with UVB rays stimulates theexpression of nitric oxide synthases-generated nitric oxide andthe formation of cytotoxic nitrogen-related oxidantsrdquoHistologyand Histopathology vol 20 no 2 pp 467ndash473 2005

[7] E Arnal C Peris-Martınez J L Menezo S Johnsen-Sorianoand F J Romero ldquoOxidative stress in keratoconusrdquo Investiga-tive Ophthalmology amp Visual Science vol 52 no 12 pp 8592ndash8597 2011

[8] R Buddi B Lin S R Atilano N C Zorapapel M C Kenneyand D J Brown ldquoEvidence of oxidative stress in human cornealdiseasesrdquo Journal of Histochemistry and Cytochemistry vol 50no 3 pp 341ndash351 2002

[9] L Zhou S Sawaguchi S S Twining J Sugar R S Federand B Y J T Yue ldquoExpression of degradative enzymes andprotease inhibitors in corneas with keratoconusrdquo InvestigativeOphthalmology and Visual Science vol 39 no 7 pp 1117ndash11241998

[10] A Valle-Prieto and P A Conget ldquoHuman mesenchymal stemcells efficiently manage oxidative stressrdquo Stem Cells and Devel-opment vol 19 no 12 pp 1885ndash1893 2010

[11] Y T Chen C K Sun Y C Lin et al ldquoAdipose-derivedmesenchymal stem cell protects kidneys against ischemia-reperfusion injury through suppressing oxidative stress andinflammatory reactionrdquo Journal of Translation Medicine vol 9no 1 article 51 2011

[12] R Dey K Kemp E Gray C Rice N Scolding and AWilkins ldquoHumanmesenchymal stemcells increase anti-oxidantdefences in cells derived from patients with friedreichrsquos ataxiardquoCerebellum vol 11 no 4 pp 861ndash871 2012

[13] H Liu S J McTaggart D W Johnson and G C GobeldquoOriginal article anti-oxidant pathways are stimulated by mes-enchymal stromal cells in renal repair after ischemic injuryrdquoCytotherapy vol 14 no 2 pp 162ndash172 2012

[14] K Kemp E Gray E Mallam N Scolding and A WilkinsldquoInflammatory cytokine induced regulation of superoxide dis-mutase 3 expression by human mesenchymal stem cellsrdquo StemCell Reviews and Reports vol 6 no 4 pp 548ndash559 2010

[15] H Nightingale K Kemp E Gray et al ldquoChanges in expressionof the antioxidant enzyme SOD3 occur upon differentiation ofhuman bone marrow-derived mesenchymal stem cells in vitrordquoStem Cells and Development vol 21 no 11 pp 2026ndash2035 2012

[16] M Krulova K Pokorna A Lencova et al ldquoA rapid separationof two distinct populations of corneal epithelial cells withlimbal stem cell characteristicsrdquo Investigative Ophthalmologyand Visual Sciences vol 49 no 9 pp 3903ndash3908 2008

[17] E SvobodovaM Krulova A Zajicova et al ldquoThe role ofmousemesenchymal stem cells in differentiation of naive T-cells intoanti-inflammatory regulatory T-cell or proinflammatory helperT-cell 17 populationrdquo Stem Cells and Development vol 21 no 6pp 901ndash910 2012

[18] V Holan P Trosan C Cejka et al ldquoA comparative study ofthe therapeutic potential of mesenchymal stem cells and limbalepithelial stem cells for ocular surface reconstructionrdquo StemCells Translational Medicine vol 4 no 9 pp 1052ndash1063 2015

[19] V Holan M Chudickova P Trosan et al ldquoCyclosporine A-loaded and stem cell-seeded electrospun nanofibers for cell-based therapy and local immunosuppressionrdquo Journal of Con-trolled Release vol 156 no 3 pp 406ndash412 2011

[20] C Cejka J Luyckx and J Cejkova ldquoCentral corneal thicknessconsidered an index of corneal hydration of the UVB irradiatedrabbit cornea as influenced by UVB absorberrdquo PhysiologicalResearch vol 61 no 3 pp 299ndash306 2012

[21] P Trosan E Svobodova M Chudickova M Krulova AZajicova and V Holan ldquoThe key role of insulin-like growthfactor i in limbal stem cell differentiation and the cornealwound-healing processrdquo Stem Cells and Development vol 21no 18 pp 3341ndash3350 2012

[22] A Zajicova K Pokorna A Lencova et al ldquoTreatment of ocularsurface injuries by limbal and mesenchymal stem cells growingon nanofiber scaffoldsrdquo Cell Transplantation vol 19 no 10 pp1281ndash1290 2010

[23] C H Lee A Singla and Y Lee ldquoBiomedical applications ofcollagenrdquo International Journal of Pharmaceutics vol 221 no1-2 pp 1ndash22 2001

[24] H S Dua ldquoAmniotic membrane transplantationrdquo British Jour-nal of Ophthalmology vol 83 no 6 pp 748ndash752 1999

[25] J Cejkova S Stipek J Crkovska et al ldquoUV rays the proox-idanhtantioxidant imbalance in the cornea and oxidative eyedamagerdquo Physiological Research vol 53 no 1 pp 1ndash10 2004

[26] E C Kim W S Lee and M S Kim ldquoThe inhibitory effects ofbevacizumab eye drops on NGF expression and corneal woundhealing in ratsrdquo Investigative Ophthalmology and Visual Sciencevol 51 no 9 pp 4569ndash4573 2010

[27] K Kemp K Hares E Mallam K J Heesom N Scoldingand A Wilkins ldquoMesenchymal stem cell-secreted superoxidedismutase promotes cerebellar neuronal survivalrdquo Journal ofNeurochemistry vol 114 no 6 pp 1569ndash1580 2010

[28] J Ueda M E Starr H Takahashi et al ldquoDecreased pulmonaryextracellular superoxide dismutase during systemic inflamma-tionrdquo Free Radical Biology and Medicine vol 45 no 6 pp 897ndash904 2008

[29] H E Lob P J Marvar T J Guzik et al ldquoInduction of hyper-tension and peripheral inflammation by reduction of extra-cellular superoxide dismutase in the central nervous systemrdquoHypertension vol 55 no 2 pp 277ndash283 2010

[30] J J Yoon S Ismail and T Sherwin ldquoLimbal stem cells centralconcepts of corneal epithelial homeostasisrdquo World Journal ofStem Cells vol 6 no 4 pp 391ndash403 2014

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


suppressed intracorneal inflammation and corneal neovascu-larization [2] It is well known that MSCs possess antioxidantproperties [2 10ndash13] The beneficial antioxidant effects ofMSCs may be explained by the findings that MSCs secretesuperoxide dismutase [14] Superoxide dismutase belongsto antioxidant enzymes that dismute toxic free radicalsproduced during inflammation [15]

To our knowledge this is the first paper investigatingthe effects of bone marrow MSCs (BM-MSCs) adiposetissue MSCs (Ad-MSCs) or corneal limbal epithelial stemcells (LSCs) on the antioxidant protective mechanism inthe regenerated corneal epithelium and on corneal opticalproperties evaluated by central corneal thickness (taken asan index of corneal hydration) and corneal transparency Wefound that in contrast to the effect of nanofibers alone (MSCs-free) where profound enzymatic antioxidantprooxidantimbalance was found in the corneal epithelium after thetreatment of injured corneas with BM-MSCs nanofibersor LSCs nanofibers (less after Ad-MSCs nanofibers) theenzymatic antioxidant protective mechanism was restored inthe regenerated corneal epithelium This was accompaniedby the renewal of corneal optical properties and acceleratedcorneal healing

2 Material and Methods

21 Preparation of StemCells LSCs were obtained by enzymedigestion of rabbit limbal tissue as we have described ina mouse model [16] In brief limbal tissue was cut withscissors and subjected to 10 short (10min each) trypsinizationcycles The released cells were harvested after each cyclecentrifuged (8 minutes at 250 g) and resuspended in RPMI1640 medium (Sigma St Louis MO) containing 10 fetalcalf serum (FCS Sigma) antibiotics (100UmL of penicillin100 120583gmL of streptomycin) and 10mM HEPES buffer Thecells were seeded in 25 cm2 tissue culture flasks (CorningSchiphol-Rijk Netherlands) For characterization of cells andfor their transfer to the nanofiber scaffold the cells growingin vitro for 2-3 weeks (3rd passage) were used

BM-MSCs were isolated from femurs of rabbitsThe bonemarrowwas flushed out a single cell suspensionwas preparedby homogenization and the cells were seeded at a concen-tration of 4 times 106 cellsmL in Dulbeccorsquos modified Eaglersquosmedium (DMEM Sigma) containing 10 FCS antibiotics(100UmL of penicillin 100mgmL of streptomycin) and10mMHEPES in 25 cm2 tissue culture flasks (Corning) Aftera 48-hour incubation the nonadherent cells were washed outand the adherent cells were cultured with a regular exchangeof the medium and passaging of the cells to maintain theiroptimal concentrationThe cells were characterized and usedafter the 3rd passage

Ad-MSCs were isolated from subcutaneous adipose tis-sue The tissue was cut into small pieces with scissors andincubated in 1mL of Hankrsquos balanced salt solution containing10mgmLof collagenase type I (Sigma) for 60minutes at 37∘Cwith gentle agitation Then the collagenase was diluted withcomplete DMEM The cells were filtered and centrifuged at250 g for 8 minutes The upper adipose layer was removedand cells were centrifuged resuspended in 6mL of complete

DMEM(4times 106 cellsmL) and seeded in 25 cm2 tissue cultureflasks (Corning) After incubation for 48 hours the cells werewashed with a medium to remove nonadherent cells and celldebris and cultured under standard conditions Ad-MSCswere used in passages 3-4

22 StemCell Differentiation andGene Expression Theabilityof stem cells to differentiate into other cell types was deter-mined according to the ability to differentiate into adipocytesusing specific adipogenic medium containing 01 120583M dex-amethasone 05mM 3-isobutyl-1-methylxanthine 01mMindomethacin and 05 120583gmL of insulin as we describedpreviously [17] Differentiation of cells was confirmed bystaining with Oil Red O

The expression of genes for the immunoregulatorymolecules indoleamine-23-dioxygenase (IDO) cyclooxy-genase-2 (Cox-2) and transforming growth factor-120573 (TGF-120573) was determined in unstimulated and lipopolysaccharide-(LPS-) stimulated MSCs and LSCs In these experiments thecells (4 times 104 cellswell) were cultured in 700120583L of DMEMfor 48 hours in 24-well tissue culture plates (Corning) with orwithout 5 120583gmL of LPS and the expression of the genes wasdetermined by real-timePCR aswe have described elsewhere[18]

23 Nanofiber Scaffold Preparation The PLA polymer waspurchased from Nature Works LLC (Minnetonka Min-neapolis Minnesota USA) This material was dissolved inchloroform at 7weight percent (wt) and two other solvents12-dichloroethane (29wt) and ethyl acetate (10 wt) (bothpurchased from PENTA Prague Czech Republic) wereadded to this solution The mixture was stirred until ahomogenous polymer solution was obtained The modifiedneedleless Nanospider technology in which polymeric jetsare spontaneously formed from liquid surfaces on a rotatingspinning electrode was used to prepare the nanofibersNanofiber scaffolds were cut into squares (approximately 15times 15 cm) and fixed into CellCrown24 inserts (Scaffdex) Theinserts with nanofibers were transferred into 24-well tissueculture plates (Corning) Three times 105 stem cells in a volumeof 700120583L of complete DMEMwere transferred into eachwellThe plates were incubated for 24 hours to allow cells to adhereto the scaffold [19]

24 Alkali Injury of the Cornea in Experimental Animals andNano Scaffold Transfer Adult female New Zealand whiterabbits (25ndash30 kg) were used in our experiments The inves-tigation was conducted according to the ARVO Statementon the Use of Animals in Ophthalmic and Vision ResearchRabbits were anesthetized by an intramuscular injection ofRometar (xylazinum hydrochloricum Spofa Prague CR2 02mLkg body weight) and Narkamon (ketaminumhydrochloricum Spofa 5 1mLkg body weight)

Sodium hydroxide (025N NaOH) was applied by meansof dropping on the corneal surface (10 drops during 1min)then the eyes were immediately rinsed with tap water Theanimals were divided into five groups (each group containedsix rabbits) In the first group of animals the injured eyeswere












(a)

0

1000

2000

3000

4000

mdashLPS

IDO

Relat

ive g

ene e

xpre

ssio

n

Relat

ive g

ene e

xpre

ssio

n

Relat

ive g

ene e

xpre

ssio

n

0

100

200

300

400 Cox-2

0

100

200

300

400

500


TGF-120573

(b)




3 Results





Nan

o

SOD

(a)

XOX

(b)BM

-MSC

s

(c) (d)

Ad-M

SCs

(e) (f)

LSCs

(g) (h)

Con

trol

(i) (j)





MMP9

Unt

reat

ed(a)

iNOS

(b)N

ano

(c) (d)

BM-M

SCs

(e) (f)

Ad-M

SCs

(g) (h)

LSCs

(i) (j)

Con

trol

(k) (l)

Figure 3 Continued


iNOS

(m)

Con

trol

Unt

reat

ed

Nan

o

BM-M

SCs

Ad-M

SCs

LSCs

0

10

20

30

lowast

lowast

Relat

ive g

ene e

xpre

ssio








Unt

reat

ed

-SMA

(a)

-SMA

Nan

o

(b)BM

-MSC

s

(c)

Ad-M

SCs

(d)

LSCs

(e)

Con

trol

(f)

0

200

400

600

800

lowast

lowast

Relat

ive g

ene e

xpre

ssio

n

Con

trol

Unt

reat

ed

Nan

o

BM-M

SCs

Ad-M

SCs

LSCs

(g)

TGF-1205731

120572120572




4 Discussion



Unt

reat

ed

VEGF

(a)

VEGF

Nan

o

(b)BM

-MSC

s

(c)

Ad-M

SCs

(d)

LSCs

(e)

Con

trol

(f)

VEGF

0

50

100

150

200

250

lowast

lowastlowast

Con

trol

Unt

reat

ed

Nan

o

BM-M

SCs

Ad-M

SCs

LSCs

Relat

ive g

ene e

xpre

ssio

n

(g)





Con

trol

(a)

Alk

ali-b

urne

d

(b)

Nan

o su

ture

d

(c)

Unt

reat

ed

(d)

Nan

o

(e)

BM-M

SCs

(f)

Ad-M

SCs

(g)

LSCs

(h)

Con

trol

Unt

reat

ed

Nan

o

Nan

o BM

-MSC

s

Nan

o LS

Cs

Nan

o Ad

-MSC

s0

10

20

30


lowastlowastlowastN

umbe

r of v

esse

ls (s

ecto

r)

(i)





0 5 150

200

400

600

800

1000








Days after injury

Cen

tral

corn

eal t

hick

ness

(120583m

)





5 Conclusions


Abbreviations




Acknowledgments


References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























(a)

0

1000

2000

3000

4000

mdashLPS

IDO

Relat

ive g

ene e

xpre

ssio

n

Relat

ive g

ene e

xpre

ssio

n

Relat

ive g

ene e

xpre

ssio

n

0

100

200

300

400 Cox-2

0

100

200

300

400

500


TGF-120573

(b)




3 Results





Nan

o

SOD

(a)

XOX

(b)BM

-MSC

s

(c) (d)

Ad-M

SCs

(e) (f)

LSCs

(g) (h)

Con

trol

(i) (j)





MMP9

Unt

reat

ed(a)

iNOS

(b)N

ano

(c) (d)

BM-M

SCs

(e) (f)

Ad-M

SCs

(g) (h)

LSCs

(i) (j)

Con

trol

(k) (l)

Figure 3 Continued


iNOS

(m)

Con

trol

Unt

reat

ed

Nan

o

BM-M

SCs

Ad-M

SCs

LSCs

0

10

20

30

lowast

lowast

Relat

ive g

ene e

xpre

ssio








Unt

reat

ed

-SMA

(a)

-SMA

Nan

o

(b)BM

-MSC

s

(c)

Ad-M

SCs

(d)

LSCs

(e)

Con

trol

(f)

0

200

400

600

800

lowast

lowast

Relat

ive g

ene e

xpre

ssio

n

Con

trol

Unt

reat

ed

Nan

o

BM-M

SCs

Ad-M

SCs

LSCs

(g)

TGF-1205731

120572120572




4 Discussion



Unt

reat

ed

VEGF

(a)

VEGF

Nan

o

(b)BM

-MSC

s

(c)

Ad-M

SCs

(d)

LSCs

(e)

Con

trol

(f)

VEGF

0

50

100

150

200

250

lowast

lowastlowast

Con

trol

Unt

reat

ed

Nan

o

BM-M

SCs

Ad-M

SCs

LSCs

Relat

ive g

ene e

xpre

ssio

n

(g)





Con

trol

(a)

Alk

ali-b

urne

d

(b)

Nan

o su

ture

d

(c)

Unt

reat

ed

(d)

Nan

o

(e)

BM-M

SCs

(f)

Ad-M

SCs

(g)

LSCs

(h)

Con

trol

Unt

reat

ed

Nan

o

Nan

o BM

-MSC

s

Nan

o LS

Cs

Nan

o Ad

-MSC

s0

10

20

30


lowastlowastlowastN

umbe

r of v

esse

ls (s

ecto

r)

(i)





0 5 150

200

400

600

800

1000








Days after injury

Cen

tral

corn

eal t

hick

ness

(120583m

)





5 Conclusions


Abbreviations




Acknowledgments


References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Nan

o

SOD

(a)

XOX

(b)BM

-MSC

s

(c) (d)

Ad-M

SCs

(e) (f)

LSCs

(g) (h)

Con

trol

(i) (j)





MMP9

Unt

reat

ed(a)

iNOS

(b)N

ano

(c) (d)

BM-M

SCs

(e) (f)

Ad-M

SCs

(g) (h)

LSCs

(i) (j)

Con

trol

(k) (l)

Figure 3 Continued


iNOS

(m)

Con

trol

Unt

reat

ed

Nan

o

BM-M

SCs

Ad-M

SCs

LSCs

0

10

20

30

lowast

lowast

Relat

ive g

ene e

xpre

ssio








Unt

reat

ed

-SMA

(a)

-SMA

Nan

o

(b)BM

-MSC

s

(c)

Ad-M

SCs

(d)

LSCs

(e)

Con

trol

(f)

0

200

400

600

800

lowast

lowast

Relat

ive g

ene e

xpre

ssio

n

Con

trol

Unt

reat

ed

Nan

o

BM-M

SCs

Ad-M

SCs

LSCs

(g)

TGF-1205731

120572120572




4 Discussion



Unt

reat

ed

VEGF

(a)

VEGF

Nan

o

(b)BM

-MSC

s

(c)

Ad-M

SCs

(d)

LSCs

(e)

Con

trol

(f)

VEGF

0

50

100

150

200

250

lowast

lowastlowast

Con

trol

Unt

reat

ed

Nan

o

BM-M

SCs

Ad-M

SCs

LSCs

Relat

ive g

ene e

xpre

ssio

n

(g)





Con

trol

(a)

Alk

ali-b

urne

d

(b)

Nan

o su

ture

d

(c)

Unt

reat

ed

(d)

Nan

o

(e)

BM-M

SCs

(f)

Ad-M

SCs

(g)

LSCs

(h)

Con

trol

Unt

reat

ed

Nan

o

Nan

o BM

-MSC

s

Nan

o LS

Cs

Nan

o Ad

-MSC

s0

10

20

30


lowastlowastlowastN

umbe

r of v

esse

ls (s

ecto

r)

(i)





0 5 150

200

400

600

800

1000








Days after injury

Cen

tral

corn

eal t

hick

ness

(120583m

)





5 Conclusions


Abbreviations




Acknowledgments


References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















MMP9

Unt

reat

ed(a)

iNOS

(b)N

ano

(c) (d)

BM-M

SCs

(e) (f)

Ad-M

SCs

(g) (h)

LSCs

(i) (j)

Con

trol

(k) (l)

Figure 3 Continued


iNOS

(m)

Con

trol

Unt

reat

ed

Nan

o

BM-M

SCs

Ad-M

SCs

LSCs

0

10

20

30

lowast

lowast

Relat

ive g

ene e

xpre

ssio








Unt

reat

ed

-SMA

(a)

-SMA

Nan

o

(b)BM

-MSC

s

(c)

Ad-M

SCs

(d)

LSCs

(e)

Con

trol

(f)

0

200

400

600

800

lowast

lowast

Relat

ive g

ene e

xpre

ssio

n

Con

trol

Unt

reat

ed

Nan

o

BM-M

SCs

Ad-M

SCs

LSCs

(g)

TGF-1205731

120572120572




4 Discussion



Unt

reat

ed

VEGF

(a)

VEGF

Nan

o

(b)BM

-MSC

s

(c)

Ad-M

SCs

(d)

LSCs

(e)

Con

trol

(f)

VEGF

0

50

100

150

200

250

lowast

lowastlowast

Con

trol

Unt

reat

ed

Nan

o

BM-M

SCs

Ad-M

SCs

LSCs

Relat

ive g

ene e

xpre

ssio

n

(g)





Con

trol

(a)

Alk

ali-b

urne

d

(b)

Nan

o su

ture

d

(c)

Unt

reat

ed

(d)

Nan

o

(e)

BM-M

SCs

(f)

Ad-M

SCs

(g)

LSCs

(h)

Con

trol

Unt

reat

ed

Nan

o

Nan

o BM

-MSC

s

Nan

o LS

Cs

Nan

o Ad

-MSC

s0

10

20

30


lowastlowastlowastN

umbe

r of v

esse

ls (s

ecto

r)

(i)





0 5 150

200

400

600

800

1000








Days after injury

Cen

tral

corn

eal t

hick

ness

(120583m

)





5 Conclusions


Abbreviations




Acknowledgments


References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















iNOS

(m)

Con

trol

Unt

reat

ed

Nan

o

BM-M

SCs

Ad-M

SCs

LSCs

0

10

20

30

lowast

lowast

Relat

ive g

ene e

xpre

ssio








Unt

reat

ed

-SMA

(a)

-SMA

Nan

o

(b)BM

-MSC

s

(c)

Ad-M

SCs

(d)

LSCs

(e)

Con

trol

(f)

0

200

400

600

800

lowast

lowast

Relat

ive g

ene e

xpre

ssio

n

Con

trol

Unt

reat

ed

Nan

o

BM-M

SCs

Ad-M

SCs

LSCs

(g)

TGF-1205731

120572120572




4 Discussion



Unt

reat

ed

VEGF

(a)

VEGF

Nan

o

(b)BM

-MSC

s

(c)

Ad-M

SCs

(d)

LSCs

(e)

Con

trol

(f)

VEGF

0

50

100

150

200

250

lowast

lowastlowast

Con

trol

Unt

reat

ed

Nan

o

BM-M

SCs

Ad-M

SCs

LSCs

Relat

ive g

ene e

xpre

ssio

n

(g)





Con

trol

(a)

Alk

ali-b

urne

d

(b)

Nan

o su

ture

d

(c)

Unt

reat

ed

(d)

Nan

o

(e)

BM-M

SCs

(f)

Ad-M

SCs

(g)

LSCs

(h)

Con

trol

Unt

reat

ed

Nan

o

Nan

o BM

-MSC

s

Nan

o LS

Cs

Nan

o Ad

-MSC

s0

10

20

30


lowastlowastlowastN

umbe

r of v

esse

ls (s

ecto

r)

(i)





0 5 150

200

400

600

800

1000








Days after injury

Cen

tral

corn

eal t

hick

ness

(120583m

)





5 Conclusions


Abbreviations




Acknowledgments


References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Unt

reat

ed

-SMA

(a)

-SMA

Nan

o

(b)BM

-MSC

s

(c)

Ad-M

SCs

(d)

LSCs

(e)

Con

trol

(f)

0

200

400

600

800

lowast

lowast

Relat

ive g

ene e

xpre

ssio

n

Con

trol

Unt

reat

ed

Nan

o

BM-M

SCs

Ad-M

SCs

LSCs

(g)

TGF-1205731

120572120572




4 Discussion



Unt

reat

ed

VEGF

(a)

VEGF

Nan

o

(b)BM

-MSC

s

(c)

Ad-M

SCs

(d)

LSCs

(e)

Con

trol

(f)

VEGF

0

50

100

150

200

250

lowast

lowastlowast

Con

trol

Unt

reat

ed

Nan

o

BM-M

SCs

Ad-M

SCs

LSCs

Relat

ive g

ene e

xpre

ssio

n

(g)





Con

trol

(a)

Alk

ali-b

urne

d

(b)

Nan

o su

ture

d

(c)

Unt

reat

ed

(d)

Nan

o

(e)

BM-M

SCs

(f)

Ad-M

SCs

(g)

LSCs

(h)

Con

trol

Unt

reat

ed

Nan

o

Nan

o BM

-MSC

s

Nan

o LS

Cs

Nan

o Ad

-MSC

s0

10

20

30


lowastlowastlowastN

umbe

r of v

esse

ls (s

ecto

r)

(i)





0 5 150

200

400

600

800

1000








Days after injury

Cen

tral

corn

eal t

hick

ness

(120583m

)





5 Conclusions


Abbreviations




Acknowledgments


References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Unt

reat

ed

VEGF

(a)

VEGF

Nan

o

(b)BM

-MSC

s

(c)

Ad-M

SCs

(d)

LSCs

(e)

Con

trol

(f)

VEGF

0

50

100

150

200

250

lowast

lowastlowast

Con

trol

Unt

reat

ed

Nan

o

BM-M

SCs

Ad-M

SCs

LSCs

Relat

ive g

ene e

xpre

ssio

n

(g)





Con

trol

(a)

Alk

ali-b

urne

d

(b)

Nan

o su

ture

d

(c)

Unt

reat

ed

(d)

Nan

o

(e)

BM-M

SCs

(f)

Ad-M

SCs

(g)

LSCs

(h)

Con

trol

Unt

reat

ed

Nan

o

Nan

o BM

-MSC

s

Nan

o LS

Cs

Nan

o Ad

-MSC

s0

10

20

30


lowastlowastlowastN

umbe

r of v

esse

ls (s

ecto

r)

(i)





0 5 150

200

400

600

800

1000








Days after injury

Cen

tral

corn

eal t

hick

ness

(120583m

)





5 Conclusions


Abbreviations




Acknowledgments


References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Con

trol

(a)

Alk

ali-b

urne

d

(b)

Nan

o su

ture

d

(c)

Unt

reat

ed

(d)

Nan

o

(e)

BM-M

SCs

(f)

Ad-M

SCs

(g)

LSCs

(h)

Con

trol

Unt

reat

ed

Nan

o

Nan

o BM

-MSC

s

Nan

o LS

Cs

Nan

o Ad

-MSC

s0

10

20

30


lowastlowastlowastN

umbe

r of v

esse

ls (s

ecto

r)

(i)





0 5 150

200

400

600

800

1000








Days after injury

Cen

tral

corn

eal t

hick

ness

(120583m

)





5 Conclusions


Abbreviations




Acknowledgments


References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0 5 150

200

400

600

800

1000








Days after injury

Cen

tral

corn

eal t

hick

ness

(120583m

)





5 Conclusions


Abbreviations




Acknowledgments


References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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