indian institute of technology delhi - modulation of macrophage...

Modulation of Macrophage Phenotype, Maturation, and GraftIntegration through Chondroitin Sulfate Cross-Linking toDecellularized CorneaJuhi Chakraborty,†,‡ Subhadeep Roy,†,‡ Sumit Murab,‡ Raghav Ravani,§ Kulwinder Kaur,‡ Saranya Devi,§

Divya Singh,§ Shubhangini Sharma,⊥ Sujata Mohanty,⊥ Amit Kumar Dinda,∥ Radhika Tandon,§

and Sourabh Ghosh*,‡

‡Regenerative Engineering Laboratory, Department of Textile Technology, Indian Institute of Technology Delhi, New Delhi 110016,India§Dr Rajendra Prasad Centre for Ophthalmic Sciences, ⊥Stem Cell Facility, and ∥Department of Pathology, All India Institute ofMedical Sciences, New Delhi 110016, India

*S Supporting Information

ABSTRACT: Decellularized corneas obtained from other species have gainedintense popularity in the field of tissue engineering due to its role to serve as analternative to the limited availability of high-quality donor tissues. However, thedecellularized cornea is found to evoke an immune response inspite of theremoval of the cellular contents and antigens due to the distortion of thecollagen fibrils that exposes certain antigenic sites, which often lead to graftrejection. Therefore, in this study we tested the hypothesis that cross-linkingthe decellularized corneas with chondroitin sulfate may help in restoring thedistorted conformationation changes of fibrous matrix and thus help inreducing the occurrence of graft rejection. Cross-linking of the decellularizedcornea with oxidized chondroitin sulfate was validated by ATR-FTIR analysis.An in vitro immune response study involving healthy monocytes anddifferentiated macrophages with their surface marker analysis by pHrodo red,Lysotracker red, ER tracker, and CD63, LAMP-2 antibodies confirmed that thecross-linked decellularized matrices elicited the least immune response compared to the decellularized ones. We implanted threesets of corneal scaffolds obtained from goat, i.e., native, decellularized, and decellularized corneas conjugated with chondroitinsulfate into the rabbit stroma. Histology analysis, three months after implantation into the rabbit corneal stromal region,confirmed the restoration of the collagen fibril conformation and the migration of cells to the implanted constructs, affirmingproper graft integration. Hence we conclude that the chondroitin sulfate cross-linked decellularized corneal matrix may serve asan efficient alternative to the allograft and human cadaveric corneas.

KEYWORDS: decellularized corneas, tissue engineering

■ INTRODUCTION

The cornea is a key component in the optical path of the eyeand serves as a transparent physical barrier to the outerenvironment. Corneal diseases are one of the major causes ofblindness that affects around 4 million people worldwide.1

Globally, there are 1.5 million cases of corneal blindnessinvestigated, whereas in India itself approximately 6.8 millionpeople suffer from vision loss at least in one eye due to cornealdiseases, which rises by 25 000−30 000 cases every year.2

Inspite of such an alarming situation, only an estimated 120 000corneal transplants are undertaken annually.3 Presently,keratoprostheses and transplantation of cornea from deceasedare primarily used for visual recovery.4 However, thesestrategies often lead to glaucoma, infection, calcification, retinaldetachment, corneal melting, and prosthesis extrusion.5 Theconstrained availability of high quality donor tissue in many

countries, standard therapy of immunosuppressive steroids aftergrafting and the rapid graft rejection in some patients have ledto the evolution of tissue engineered corneas as a potentialsubstitute to traditional corneal grafts.In an attempt to fabricate tissue engineered cornea, corneal

cells have been cultured into wide varieties of natural orsynthetic polymeric scaffolds, for example, recombinant humancollagen hydrogel and recombinant human collagen 2-methacryloylxyethyl phosphorylcholine,6 silk fibroin,7,8 gelatinhydrogel,9 gelatin-polyHEMA,10 etc. Invariably it is impossibleto replicate the precise anatomical microstructure and

Special Issue: Biomaterials Science and Engineering in India

Received: March 2, 2018Accepted: May 1, 2018Published: May 1, 2018

Article

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© XXXX American Chemical Society A DOI: 10.1021/acsbiomaterials.8b00251ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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extracellular matrix (ECM) composition of the cornea.Presence of random pores in the polymeric scaffolds makes itdifficult to control cellular orientation, achieve adequatestrength and transparency, which are the major requirementsin generating a functional cornea.11 Corneal transparency isgoverned by the orderly arrangement of collagen fibers in thestroma. The collagen type I and collagen type V fibers in thestroma, which are usually 25−30 nm in diameter, are organizedin a parallel fashion with a regular spacing of 30 nm betweeneach fibril. This precisely regular spacing is controlled by thepresence of the proteoglycans around the collagen fibrils. Thisorderly array of fibers along with the proteins expressed by thekeratocyte cells are known to play a significant role in cornealtransparency.12 Degradation of the implanted biomaterials(cross-linked recombinant human collagen), haze at theinterface between the host stroma and the graft, delayedepithelial closure caused postsurgical astigmatism, and highestcorrected visual acuity of 0.4 could be achieved in six out of tenpatients at 24 months.13 A four year long, phase-1 humanclinical trial was conducted to replicate the corneal structureusing cross-linked recombinant human collagen type III and 2-methacryloyloxyethyl phosphorylcholine, a synthetic phospho-lipid, as a corneal substitute in patients who were at high risk ofgraft failure. This strategy was able to restore vision in two outof three patients.14 Therefore, the requirement of a moresuitable biomaterial that can be easily colonized by host cornealcells, integrate with surrounding tissue, and restore the cornealtransparency is a prerequisite in fabrication of a functionalcornea.As the polymeric scaffolds fail to replicate the native corneal

ECM chemistry and architecture, corneas from other speciessuch as pig, rabbit, sheep may serve as an alternative to humancorneal tissue.15 The corneal allograft transplantation isexpected to be successful because of the immune privilegednature of the corneal tissue.16 The reason for this immuneprivilegeness can be attributed to the absence of blood vessels,lymphatic system in the cornea, the existence of immunomo-dulatory factors in the aqueous humor, and the blood/eyebarrier. As a result, the time taken between the cornealtransplantation and antigen recognition gets extended, thusarresting the entrance of the effector cells.17 In addition, lowlevels of MHC class I molecule expression and no expression ofMHC class II molecule at all by the corneal epithelial,endothelial, and keratocyte cells result in immune privilege.18

However, there is a gap in the proper understanding of themechanism of immune privilege, as corneal transplantationoccasionally ends up with inflammation and corneal allograftrejection.19

Decellularization of the corneas from other species(xenografts), from which the cells and antigen molecules areeliminated to decrease the host immune reaction, have gainedincreasing interest and are suggested to serve as a promisingalternative to human cadaveric corneas. Lately, several groupshave demonstrated decellularization of cornea by using aplethora of methods, including chemical means by using Trisbuffer, ethylene diamine tetraacetic acid and sodium dodecylsulfate,20 by using enzymes such as nucleases, phospholipaseA2, trypsin etc21,22 and physical methods such as freeze-dryingor high hydrostatic pressure.23 There is a strict requirement thatthe process of decellularization should absolutely remove allcellular components/material including the lipid membranesand antigen molecules within the tissue and membrane surface,solubilize the cytoplasmic and nuclear cellular molecules, DNA

fragments, while simultaneously removing the cellular debrisfrom the tissue, to reduce any possible host rejection orimmunological reaction. At the same time, the decellularizationprotocol must retain the structural, functional properties, andintegrity of the ECM.24 Preservation of pseudohexagonal latticestructure of collagen bundles is a crucial step while designing aprotocol for decellularization, to ensure transparency. Inaddition, the decellularized corneal matrix should be efficientin sustaining the adhesion and proliferation of the corneal cellsand therefore should act as a matrix resembling the nativecornea.25,26 In our previous study, we compared variouschemical and physical methods of decellularization, where weconcluded that the corneal decellularization using a perfusionbioreactor with 0.1% Triton-X 100 (nonionic detergent, t-octylphenoxy polyethoxyethanol) resulted in an efficientremoval of cellular and nuclear material.27 A unique findingof that study was the controlled unidirectional flow of detergentat the rate of at 10 mL min−1 directly through the corneapersuaded cells to undergo apoptosis (but not necrosis) andassisted in efficient removal of cells, causing improveddecellularization efficiency, while retaining the ECM ultra-structure. This may offer crucial implication for inflammatoryresponse post-transplantation in two ways. First, decellulariza-tion of a tissue through apoptotic pathways is a preferred modeto induce cell death compared to necrosis trajectory, as thiswould trigger phagocytosis by immune cells, and suppress theimmune reactions. But necrosis induced impaired membraneintegrity may result in inflammation and graft rejection.27

Second, in a follow up study,28 we elucidated that although thedisturbance to the ECM ultrastructure can be minimized byfollowing this optimized slow perfusion-based decellularizationstrategy, excessive hydration and disruption of secondaryconformation of the collagen stromal fibers cannot be entirelyevaded. ATR-FTIR and Raman spectroscopic analysis showedthat the α-helix to β-sheet ratios decreased significantly afterdecellularization by Triton X-100 treatment, along withincrease in random coils and β-strands content. This insightgenerates a concern about the exposure of certain antigenicsites during the process of decellularization, which areotherwise buried within the triple helical structure. These twoimportant aspects are often ignored in development ofdecellularized matrix for organ regeneration. At the sametime, partial alteration in conjugation between collagen anddecorin and keratan sulfate was noticed. Osmoregulatory agentssuch as glycerol and dextran could retrieve the nativeconformation of collagen fibrils present in the stromal layer,as well as facilitated reattachment of decorin or keratan sulfatemolecules to the collagen molecules but they were insufficientin regaining the native structural properties of the cornea.29

Decellularization induced modulation in collagen conformationwas further corroborated by Hwang et al.,30 through specificbinding of a collagen hybridizing peptide with denaturedcollagen with altered conformation. It would be interesting toinvestigate how denatured collagen fibrils with modulatedconformation at the decellularized corneal matrix may triggerinnate immune response.At the same time, strategies to conjugate proteoglycans with

collagen fibrils of decellularized ECM may help in restoringchemical composition of the stroma, while imparting specificbiological functionality.31 A major component of decorin is theglycosaminoglycan (GAG) chain, consists of chondroitinsulfate, dermatan sulfate or their copolymer. ChondroitinSulfate (CS) is a sulfated GAG made up of a chain of

ACS Biomaterials Science & Engineering Article

DOI: 10.1021/acsbiomaterials.8b00251ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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alternating glucoronic and N-acetyl galactosamine sugars.28

Cross-linking CS with N-hydroxysuccinimide (NHS) groupresulted in an increase in the diameter and density of thecollagen fibers and also improved the mechanical strength ofthe cornea in Keratoconus rabbit models.32 In addition, CS wasknown to play a significant role in wound healing.28 CS isreported to play a crucial role in reducing immune response32

and at the same time known to increase mechanical strength.33

Inflammatory responses in many tissues are tightly regulated byactivation of NF-kB transcription factors. CS exerts anti-inflammatory activity by inhibiting the nuclear translocation ofnuclear factor κ-light-chain-enhancer (NF-κB) of activatedimmune cells and have a stabilization effect on the DNAbinding domain (NF-kBp65) by regulating its deacetylation.34

Because it is an essential component of the ECM of manytissues including the cornea, we hypothesized that CSconjugation with decellularized corneal stromal ECM wouldresult in enhanced graft integration, reduced foreign bodyreaction, or innate immune response and tissue inflammation.Therefore, in the present study we attempted to address two

questions. First, whether native or decellularized corneas wouldinflict immune response when implanted from one species(goat) to other (rabbit). Our second aim was to find how cross-linking the decellularized corneas with CS would modulate theimmune response, both in vitro and in vivo. Thus, we aimed toreduce the immune response of the decellularized cornea bycross-linking it with CS that would result in increasing itsbiomechanical strength by replenishing the proteoglycan lossand recovering its biochemical composition. To the best of ourknowledge this is the first study on the use of CS for the cross-linking of decellularized goat corneas as an alternative methodfor generating corneal substitute for transplantation. We testedthe potential of our decellularized cross-linked cornealconstructs from goat as a corneal graft for transplantationinto rabbit corneal stroma and validated this both in vitro andin vivo by studying the comparative effect of immune responsegeneration using three sets of corneal scaffold (native cornea,NC; decellularized cornea, DC; and decellularized cornea cross-linked to oxidized chondroitin sulfate, DC+CS).

■ MATERIALS AND METHODSCornea Isolation. Cadaveric goat eyes were collected from All

India Institute of Medical Sciences, New Delhi, with prior approvalfrom the institute ethical committee. Corneas form the goat ocularglobe were excised under sterile conditions and washed five times inPBS containing 100 U/ml penicillin, 50 μg/mL gentamycin (Himedia,India), 100 μg/mL amphotericin B (Himedia, India) and streptomycin(Lonza, USA).Decellularization. The dissected tissue was decellularized using a

perfusion bioreactor (kindly provided by Prof. Ivan Martin, Basel,Switzerland),35 as described elsewhere.27 Briefly, Triton-X 100 (0.5%in PBS) was perfused directly through the corneal tissue using aperistaltic pump (KD Scientific, USA) unidirectionally at a constantflow rate of 50 μL/min for 48 h. Thereafter, the decellularized tissuewas rinsed through the same bioreactor by perfusing antibiotic mixtureat a flow rate of 10 μL/min for 48 h.Cross-Linking with Chondroitin Sulfate (CS). CS (from bovine

trachea, MW 105 Da., Merck, Catalogue No. 6A2942) was oxidizedusing periodate salt. Briefly, 616 mg of NaIO4 and 600 mg of CS weredissolved in 10 mL of deionized water at dark. The reaction wascontinued for 1 h, with vigorous stirring at 40 °C. The reactionproduct was purified by filtration with Sephadex G-25 (Sigma) size-exclusion chromatography.36 In next step, decellularized corneas werefirst rinsed for 1 h with sodium acetate solution (10% w/v),thoroughly washed with deionized water and subjected to cross-

linking with oxidized CS (6% w/v in distilled water). Finally cross-linked decellularized corneas were thoroughly washed in deionizeddistilled water to ensure that there was no unbound CS left.

Attenuated Total Reflectance-Fourier Transform InfraredSpectroscopy (ATR-FTIR). ATR-FTIR spectrum of the freeze-driedsamples (NC, DC, DC+CS) was obtained using an Alpha-Pspectroscope (Bruker, USA). An infrared spectrum was acquired intransmittance mode in the spectral region of 400−4000 cm−1 and aspectral resolution of 4 cm−1, and 240 scans were taken for eachsample. Origin 8.5 was used for obtaining the peaks in the givenspectral region.

In Vitro Immune Response Study. Cell Culture. Humanmonocyte cell line THP-1 (derived from acute monocytic leukemia)was maintained in RPMI 1640 medium supplemented with 10% fetalbovine serum (FBS), penicillin-streptomycin, gentamicin and 2 mMsodium pyruvate. THP-1 monocytes were differentiated into macro-phages by treating with phorbol-12-miristate-13-acetate (PMA) at aconcentration of 20 ng/mL, in 6-well microplates at a concentration of5.6 × 105 cells per well in complete RPMI 1640 media.37 After 72 h,differentiated cells were washed with PBS for three times and thenseeded with the fresh culture medium with different groups of cornealscaffold (NC, DC+CS). Freshly cultured monocytes were alsoincubated with NC, DC, DC+CS corneal matrices.

Scanning Electron Microscopy. The constructs were fixed in 10%formalin for 20 min, followed by washing twice with PBS anddehydration in graded alcohol series and air drying. The samples werethen sputter coated with gold (up to 15−20 nm thickness) using anEMITECH K550X (UK) sputter coater set at 25 mA for 240 s. Thecoated samples were imaged at varied magnifications using a JEOL5610LV (JEOL; Japan) SEM at an accelerating voltage of 5 kV.

Initiation of Fluid Phase Uptake or Bulk Phase Endocytosis inTHP-1 Cells. After incubation of monocytes and differentiatedmacrophages with different corneal scaffolds, 50 ng/mL concentrationof pHrodo Red Dextran stain (Thermo Fischer Scientific, India) wasadded. After incubating the cells for a period of 1 h, the constructswere rinsed twice with 10 mM PBS and were fixed for 15 min at roomtemperature in dark with BD cytofix/cytoperm assay kit. Corneascaffolds were treated with background suppressor for respective blueand red channel to decrease signal-to-noise ratio and nonspecificlabeling and air-dried and mounted in fluorescent mounting mediumProlong Gold antifade reagent with DAPI in a one end frosted glassslide and their fluorescence was observed and recorded on invertedconfocal laser scanning microscope using Leica TCS SP5 (LeicaMicrosystems, Germany) equipped with an argon laser (457−514nm), a diode laser (405 nm), a DPSS laser (561 nm). The correctedtotal cell fluorescence was calculated as a measure of fluorescenceintensity using ImageJ (NIH).

Lysosomal and Endosomal Trafficking in THP-1 Cells. THP-1cells were seeded on corneal matrices for 72 h at 37 °C in a 5% CO2.After that they were incubated with 50 nM Lysotracker Red D99(Molecular Probes, USA) and ER Tracker Red (BODIPY, MolecularProbes, USA) at 37 °C for 2 h. Subsequently samples were washedtwice and fixed and permeabilized (BD Cytofix/Cytoperm kit, USA)for 20 min at room temperature, washed with PBS and were treatedwith Signal Enhancer for the period of 45 min, stained with DAPI(Invitrogen, Italy), and mounted with Prolong Gold antifade mountingmedium. Samples were treated with background suppressor forrespective blue and red channel to decrease signal-to-noise ratio andnonspecific labeling. The samples were viewed by confocal scanningmicroscopy using Leica TCS SP5 (Leica Microsystems, Germany)equipped with an argon laser (457−514 nm), a diode laser (405 nm),a DPSS laser (561 nm) to access the Lysotracker Red D99 and ERtracker positive region by using an oil immersion 20× objective lens.Frame averaging were done with 3−4 scan to reduce noise. Thecorrected total cell fluorescence was calculated as a measure offluorescence intensity using ImageJ (NIH).

CD63 and LAMP-2 Staining in THP-1 Cells. Cells were fixed with(BD Cytofix/Cytoperm kit, USA) for 20 min at room temperature.Before antibody staining the samples were treated with Image IT FXsignal enhancer for duration of 45 min. Cellular endogenous



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peroxidase activity has been quenched by adding 3% H2O2 into thecorneal stromal matrix. Then the samples were incubated with 100−150 μL blocking buffer (10% goat serum and 3% BSA) for a period of2 h in room temperature and processed with primary antibody againstCD63 (Origene, TA802751), LAMP-2 (Origene, TA336932). Allprimary antibodies were diluted with Blocker BSA (Thermo Fisher,554657) in a dilution of 1:500 and applied 500 μL for each samples forovernight at 4 °C. After the primary labeling, the cells were washedthree times with wash buffer and incubated with poly-HRP-conjugatedsecondary antibody for 2 h at room temperature. 100 μL of thetyramide working solution (Thermo Fisher, B40916) was added toeach sample for a period of 2−10 min. Reaction was stopped byadding 100 μL of reaction stop reagent. Tyramide conjugation(Tyramide, H2O2, Reaction buffer) was performed to get a superiorsignal from poly-HRP conjugated secondary antibody with photostablebight Alexa Fluor 555. Corneal scaffolds were washed three times withPBS and mounted with Prolong Gold antifade mounting mediumcontaining DAPI. Indirect immunofluorescence was examined using aconfocal laser microscope using Leica TCS SP5 (Leica Microsystems,Germany) equipped with an argon laser (457−514 nm), a diode laser(405 nm), a DPSS laser (561 nm). DAPI stained images wereconverted to 3D using the Leica LAS X 3D Visualization software(Leica Microsystems, Germany). The corrected total cell fluorescencewas calculated as a measure of fluorescence intensity using ImageJ(NIH).Implantation in Rabbit Models. The corneas were washed five

times in PBS (Himedia, India) containing 100 U/mL penicillin, 50 μg/mL gentamycin (Himedia, India), 100 μg/mL amphotericin B(Himedia, India) and streptomycin (Lonza, USA) and then cut fromthe middle and punctured with a biopsy punch to take the circularcorneal construct from the stroma. Adult New Zealand white rabbitswere used for the experiments. Twelve week old, two males and onefemale rabbits weighing 1.62−2.12 kg were taken for each group (n =3). The rabbits were treated following the ARVO Statement on theUse of Animals in Ophthalmic and Vision Research. All animalexperiments were approved by the institutional ethical and animalethics committee of All India Institute of Medical Sciences (AIIMS),New Delhi, India (Animal ethics clearance number 869/IAEC/15).The rabbits were anesthetized by using xylazine hydrochloride (5

mg/kg) (Indian Immunologicals Ltd., Hyderabad, India) and ketaminehydrochloride (35 mg/kg) (Trolkaa Pharmaceuticals Ltd., Dehradun,India). Left eye was implanted with the corneal implants in eachrabbit. The recipient cornea was trephinated with a 4 mm biopsypunch to make the pocket. Decellularized corneas were trephinatedusing 3 mm biopsy punches to fit them into the recipient bed. Thedonor corneal implant was trephinated using a 4 mm biopsy punchand cut thereafter. The constructs were inserted into the cornealpocket and sutured with 10−0 nylon cardinal sutures (Video S1).Topical steroid- 1% w/v Prednisolone acetate (Sun PharmaLaboratories Ltd., Mumbai, India), antibiotic- Moxifloxacin (FDCLtd. Aurangabad, India) and lubricant- 0.3% hypo-mellosebenzalko-lium chloride (Sunways India Pvt. Ltd., Ahmedabad, India) wereadministered thrice daily for 3 weeks. After three months the rabbitswere sacrificed by an overdose of sodium pentobarbital (200 mg/kg).Both the eye balls of all the rabbits were excised out of the orbits andimmersed in 4% paraformaldehyde solution for fixation.Microscopic Grading of Corneal Haze. Corneal grading system

reported by Fantes et al.38 was used for the grading of the cornealhaze: grade 0, cornea that was completely clear; grade 0.5, cornea witha slight haze by slit lamp microscopic examination; grade 1, haze that isnot interfering with the visibility and noticeable fine details of the iris;grade 2, slight obstruction of the details of the iris; grade 3, a moderateobstruction of both the lens and the fine iris details; grade 4, novisibility of the corneal details due to complete opacification of cornea.The grading of the corneal haze was done by three independentresearchers in a masked manner.Histological Analysis. Three months after the implantation, the

three groups of corneas, viz. NC, DC, and DC+CS, were harvested,fixed in 4% paraformaldehyde, and subjected to routine processing forhistological examination. Five-micrometer-thick paraffin sections were

taken for H&E staining. Subsequently, five sections for each groupwere examined under a light microscope. The images were capturedfor further analysis (Leica DFC295, Germany) using Leica softwareapplication suite (LAS V3.8). A grading scale of 0−3 was used todetermine cellular Infiltration, vascularization, inflammation, loss ofnuclei in stromal cells, and stromal cell separation. The grading systemused is

(A) Cellular infiltration & inflammation (ten consecutive highpower fields (HPF) (20×) were assessed under microscope (E600, Nikon Corporation, Japan). Grading: 0 = no cell/10 HPF,1= 1−5 cells/10 HPF, 2 = 6−10/10 HPF, 3 = >10 cells/10HPF.

(B) The grading of vascularization was done in ten consecutive highpower fields (20×) in the area of hotspot (where the vesselswere seen). Grading: 0 = no vessel/10 HPF, 1 = 1−3 vessels/10 HPF, 2 = 4−6 vessels/10 HPF, 3 = >6 vessels/10 HPF

(C) For grading of the stromal changes 50 consecutive high powerfield (20×) in the stromal region was evaluated and grading wasdone by number of field showing changes/10 HPF. Grading: 0= no stromal changes/10 HPF, 1 = Stromal changes 1−5 areas/10 HPF, 2 = 6−10 areas/10 HPF

Statistical Analysis. Data are presented as mean ± standarddeviation, with n representing the number of experiment repeated.Student’s t test was carried out to establish statistical significance andprobability at p < 0.05 was considered significant. Each experiment wasconducted with n = 3 and were repeated twice.

■ RESULTS AND DISCUSSIONCharacterization of decellularized cornea was done in terms ofresidual GAG and DNA content (Table S1), as well as absenceof cell nuclei, as reported earlier.27

Cross-Linked Corneal Ultrastructure Analysis by ATR-FTIR Spectroscopy. To understand the interaction betweenthe corneal collagen type I and the oxidized chondroitin sulfate(CS), FTIR spectrum of the studied samples were obtained(Figure 1). After cross-linking the DC with oxidized CSfollowed by washing steps, it was possible to identifycharacteristic bands of CS in the spectrum. Amide I is themost sensitive band for detecting changes in the cornealstromal collagen type I layer and its characteristic transmission

Figure 1. FTIR spectra of the stroma from native goat cornea,decellularized cornea, and chondroitin sulfate conjugated decellular-ized cornea.



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band was found to be located in the range 1600−1700 cm−1.39

The amide I peak that is centered at 1630 cm−1 corresponds tothe CO stretching vibration.40 This amide band was shiftedto lower wavenumber i.e. 1623 cm−1 in case of DC+CS sample.The observed “red shift” was due to the chemical interactionbetween the COO− and SO3

− of CS and CO of collagenfibrils. An increase in the intensity of the peak at 1623 cm−1 isdue to the overlapping of amide I and O−H vibrations withmaximum intensity. Asymmetrical stretching modes of COO−

and SO3− ions in CS were observed at 1623 and 1238 cm−1.

Amide II positioned at 1542 cm−1 corresponds to the presenceof N−H plane band and the C−H stretch vibration41 and C−Ostretching vibrations.42 Vibration at 1241 cm−1 was assigned toamide III vibration of cornea epithelial Collagen type I layer.43

Band near 1350 cm−1 was assigned to the symmetric methylbending vibrations of the acetate anion of CS. Band near 1070is due to C−O−C stretching.44,45 No peak corresponding tostructural changes of corneal collagen type I component wasfound, i.e., in the range of 1610−1620 cm−1. Band near 3300cm−1 was due to O−H vibrations in all the samples. The cross-linking time and density was optimized using a number ofcorneas and only the most optimized result has been reported.

The steps involved and the mechanism of cross-linking hasbeen explained in the experimental protocol (Figure S1).Hence, on the basis of FTIR analysis we concluded that theoxidized CS binds with CO of the corneal collagen type Ithrough COO− and SO3

− linkage. This cross-linking may helpcollagen type I layer to regain its stability after thedecellularization procedure and thus maintaining lamellarorientation akin to native cornea.46,47 Covalent tethering ofCS with collagen fibrils were reported to result in increasedfibril density and oriented lamellae when compared to fibrils inonly DC.48 CS conjugation also reported to preserve theregular collagen fibrillary dimensions compared to thedestructive corneal collagen architecture in DC.49

SEM Analysis. SEM analysis was done to determine themorphology and surface architecture of the THP-1 monocytes72 h after seeding on corneal scaffold. The monocytes (withoutPMA treatment) grown on two groups of corneal matrices (NCand DC+CS) displayed spherical morphology with nominalruffles and blebs noticeable on their surfaces. However, theTHP-1 monocytes culture on the DC scaffolds differentiatedinto macrophages, without any chemical activation, which wasevident due to increase in their cell size (103.77 μm2), feret’s

Figure 2. SEM of THP-1 monocytes seeded on different corneal scaffolds cultured for 72 h, i.e., NC (A, D), (B, E) DC, and (C, F) DC+CS withoutPMA and on (G, J) NC and (H, I, K) DC + CS with PMA. Magnifications of 2000× and 5000× of same field is used. Red arrows represent (A, D)dividing THP-1 cells, (B, E) macrophage activation, (H, J) giant cell formation.



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diameter (1.14 μm) as well as alteration in cell roundness(0.813 μm2) and solidity (0.920) as compared to the NC(significant at p < 0.05) (Figure S2). Protrusion of the flagellaor the extracellular projection was visible in many cells (Figure2B, E). However, upon treatment of NC and DC+CS withPMA, there was a loss of mononuclear shape and THP-1 cellsvisibly became more flattened after differentiation. Occasionallywe could identify the formation of giant cell macrophage whichresulted in an increase in cell size (Figure S2) due to cell fusion(Figure 2H, J).Taken together, from morphological analysis, it appeared

that native corneal matrix supported growth of the THP-1monocytes, and they were inactivated probably due its immuneprivilege nature. However, we cannot exclude the possibilitythat upon decellularization, disruption of collagen conformationin stromal layer by Triton-X treatment49 may expose the centraldeterminants (the hidden antigenic site at nonhelical terminalregion) which play a major role in collagen−antibodyinteractions, and may elicit an immune response.29,50,17 Toaddress this issue, the differentiation of the THP-1 monocytesinto macrophages was studied on the DC without any externalstimulation (Figure 2B,E). Two other sets of corneal scaffold(NC, DC+CS) would be two other control experimentalgroups. THP-1 monocytes under the influence of chemicalstimulations or upon exposure to the cornea stromal ECMcomposition may further differentiate into M2 macrophage.51

Even after stimulation of the monocytes with PMA, the DC+CS corneal scaffold showed lower extent of differentiation ofthe monocytes into macrophages compared to NC and DC,thus confirming less immunogenic response in CS-conjugatedstromal matrix.Fluorescence and Immunofluorescence Imaging Re-

sults. Initiation of Fluid Phase Uptake or Bulk PhaseEndocytosis in THP-1 Cells. Monocyte population growing onNC (Figure 3A) and DC+CS (Figure 3G) scaffold remainnegative upon pHrodo red dextran staining, whereas cells onDC (Figure 3E) and PMA stimulated group of NC (Figure 3C)and DC+CS (Figure 3I) showed bright red fluorescence fromthe acidic region. It clearly demonstrated the initiation of M2-mediated phagosomal uptake in different experimentalconditions.To study the behavior of monocytes and macrophages on

corneal scaffolds we started from the fundamental aspects ofconversion of monocyte to macrophage in the putativeimmunogenic sites. Detailed downstream study based onsurface marker analysis and macrophage maturation revealedrole of decellularized corneal matrix to trigger immuneresponse (Figure 7B). Dextran conjugate has been widelyused to monitor trafficking of different endocytic cargo whichemits bright fluorescence in accumulated active vacuolarATPase enriched acidic environment and remain unresponsiveat neutral pH. Phagocytosis and endocytosis both are verytightly regulated engulfment phenomena through which healthymacrophages can engulf particulate (microorganism, smallmolecule) and nonparticulate material (protein, polysacchar-ide). In the present study we have explored this uptakephenomenon to identify the monocyte/macrophage populationin different experimental conditions. By applying chemicalstimuli, distinct phenotypic macrophage polarization (classicalactivation, M1-like cells; alternative activation, M2-like cells)occur in THP-1 monocyte.51,52 Both of these phenotypes havethe ability to generate phagosomes, but with distinctivelyseparate pH microenvironment of coated vesicles, which

basically depends on the proposed target for vesicular uptake.Progressive luminal acidification is a hallmark of macrophagematuration as well as its later fusion with endocytic pathway.53

Figure 3. Fluorescence microscopy analysis of pHrodo red dextranstaining of THP-1 monocyte on (A, B) NC, (E, F) DC, (G, H) DC+CS, and (C, D) PMA stimulated macrophage on NC; (I, J) DC+CScultured for 72 h.Dextran red positive and merged (Dextran redpositive + DAPI) channel has been represented in 20× magnification.(K) Fluorescence intensity comparison graph. Data represented asmean ± SD, *represents p < 0.05.



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As M1-like macrophages show neutral or slightly alkaline (7.6)pH compared to the M2 subtype ( pH 4.8), we have targetedthis specific pH to distinguish the phenotypes. Higheraccumulation of dextran signal from monocytes populationsseeded upon DC group validated our first hypothesis thatdecellularized ECM composition might be able to differentiatethe monocytes into macrophages because of the exposedantigenic moieties.Lysosomal and Endosomal Trafficking in THP-1 Cells.

PMA mediated differentiated macrophage on NC and DC+CSsurface showed ER tracker red (Figure 4C, I) and Lysotrackerred D99 (Figure 5C, I) positive signal without any signal fromthe monocyte population. Whereas DC itself showed character-istics of increased lysosomal and endosomal content in respectof its ECM composition without any external chemical stimuli(Figures 4E and 5E).To confirm the fate of increased phagosomal cargo present in

the cytoplasm, we next stained for two organelles whosecytoplasmic number contributes to macrophages' granularity,namely lysosomes and endosomes, which accumulate withmacrophage differentiation. Vacuolar H-ATPase (v-ATPase)and its fusion (phagosome-lysosome) promoting role withmaintaining subcellular acidification (pH 4.5−5.0) of someorganelles is a long debated paradigm.54 Uptake of thisphagosomal cargo reaches the lysosome via the endocyticpathway in the presence of large multiprotein complex, vacuolarH-ATPase.55 Detailed investigations directed by acidotropic orweakly basic amine fluoroprobes that greatly protonated itsfluorophores attached with weak base in acidic environmentreveals the pathogenesis and biosynthesis of different acid richorganelles. Lysotracker Red D99 and ER-tracker red were usedas a pH dependent marker of lysosomal and endosomalcompartment, which is highly selective for acidic organelle. Thefate and maturations of initial vacuolar cargo, stained bypHrodo red dextran in previous experiment, now get defined byhigh intensity of lysosomal and endosomal signal from PMAstimulated macrophage (NC, DC+CS). Cells cultured over DCsurface displayed positive signal from differentiated macrophagein response to ECM without any external chemical stimulation.CD63 and LAMP-2 Staining in THP-1 Cells. PMA

stimulated macrophages on NC and DC+CS surface expressedCD63 (Figure 6E, N), LAMP-2 (Figure 7Ae, n), whereas themonocyte population reflected a decreased amount of CD63and LAMP-2 protein in the lysosomal surface. But cells grownon DC show characteristics of increased lysosomal content(Figures 6H and 7Ah), even without external stimuli, comparedto Native cornea (NC).A group of tetraspanin membrane protein CD63 and LAMP-

2, which represent two different lysosome associated membraneglycoprotein, characteristics of late endosome-phagososme andphagolysosome, respectively.55 Heavily glycosylated luminaldomain and a short cytosolic tail of CD63 form a continuouscarbohydrate lining on the inner leaflet of lysosomal membrane,generating a glycocalyx exhibit considerable sequence homol-ogy and have similar domain structure and biochemicalproperties, which maintain the integrity and provide protectionagainst luminal hostile acidic environment.56 After gettingconfirmation from Lysotracker Red D99 and ER tracker redstaining in acidic region we have targeted membrane proteinswhich are essential for lysosome maturation. Stimulatedmacrophages on NC and DC+CS surface showed intensesignal of CD63 and LAMP-2, with similar signal intensityobserved from macrophages in DC matrix.

Taken together, the early stage innate immune response ormonocyte−macrophage differentiation can be considered as a

Figure 4. Fluorescence microscopy analysis of endosomal staining ofTHP-1 monocyte on (A, B) NC, (E, F) DC, (G, H) DC+CS, andPMA-stimulated macrophage on (C, D) NC and (I, J) DC+CScultured for 72 h. ER tracker red and merged (ER tracker red + DAPI)channel has been represented in 20× magnification. (K) FluorescenceIntensity comparison graph. Data represented as mean ± SD,*represents p < 0.05.



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predicting marker for biocompatibility, clinical outcome, andchance of rejection of a biomaterial. These detailed insightsabout the monocytes/macrophage behavior and followed by its

maturations on corneal ECM paved the way to predict putativehost immunological aspect after implantation.

Microscopic Grading of Corneal Transparency. Micro-scopic examination of corneas postoperatively revealed focalhaze (grade 0.5) for first few days. But after 1 week clearcorneas were observed by microscopic examination in all thegroups with no clinically noticeable corneal haze (grade 0).

Histology Analysis. In our earlier study,27 decellularizedcornea produced by our standardized protocol of perfusing0.1% Triton-X directly through the cornea using a perfusionbioreactor was characterized by absolute absence of nuclearcontent, minimal DNA content (20 ± 0.2 ng/mg of dry wt oftissue), cellular fragments as well as membrane associatedantigens, while preserving the ECM ultrastructure. In addition,decellularization protocol led to the removal of the endotheliallayer, but ultrastructure of Descemet’s membrane and Bow-man’s membrane was preserved. Triton-X-mediated decellula-rization is believed to disrupt lipid−protein and lipid−lipidinteractions, but does not disrupt protein−protein interactionsthat is supposed to maintain ultrastructure of collagen fibril.49

Hence, the 0.1% Triton-X-treated cornea displayed bettertransparency compared to SDS and freeze−thaw-treated corneaat visible wavelength (400−800 nm range).Histology images were analyzed after 3 months of

implantation of three corneal matrices (NC, DC, DC+CS)into the rabbit cornea stromal pocket. Three months postimplantation, the distinct five layered structure found to be wellpreserved in the rabbit cornea in which stroma from native goatcornea were implanted. The epithelial layer made up ofstratified squamous epithelium, followed by the bowman’smembrane rich in elastic fibers, stromal layer made up ofparallaly arranged collagen fibers and filled with keratocytenuclei, descemet’s membrane and endothelium layer wereclearly visible (Figure 8 C, D). There was extensive cellularinfiltration of the recipient rabbit’s stromal cells in thedecellularized matrix (DC) as well as similar phenomenonwas observed in comparatively lesser extent in other groups(NC, DC+CS). The DC matrix was also found to be seamlesslyintegrated into the rabbit’s stromal layer such that it wasdifficult to distinguish the implanted tissue from the nativecornea (Figure 9). Interestingly, no inflammatory cells wereobserved in the subepithelial region (Figure 9C, Table 1) of theDC. Epithelial region downgrowth into the anterior chamberwith vascular invasion has been observed in the DC which maybe a result of initial inflammation produced by the surgicalimplantation procedure. Absence of hyperplasia in the incisionsite with irregular lamellae orientation is observed in the DC(Figure 9A−E). Extensive infiltration of stromal cells was seenin this cross-linked (DC+CS) group from the surroundingstroma. However, there was only focal disorganization of thelamella of stromal cells and no separation among the lamellanoted (Figure 10). There was seamless integration of the graftand however there was no sign of inflammation (Figure 10A−D, Table 1).57 No evidence of epithelial down growth into theanterior chamber was observed. Hyperplasia of cornealepithelium was observed near the incision site.Yoeruek et al.20 decellulared porcine corneas using 0.3%

sodium dodecyl sulfate and implanted in stromal pockets ofrabbit cornea. Major infiltration of immune competent cells wasnoticed, along with some regions of disorganized ECMarchitecture. As a result, initially they noticed prominentcorneal opacity, but after 6 months that was improved to grade1−2. Hasimoto et al.58 decellularized porcine cornea using high

Figure 5. Fluorescence microscopy analysis of lysosomal staining ofTHP-1 monocyte on (A, B) NC, (E, F) DC, (GH) DC+CS, andPMA-stimulated macrophage on (C, D) NC and (I, J) DC+CScultured for 72 h. Lysotracker red and merged (Lysotracker red +DAPI) channel has been represented in 20× magnification. (K)Fluorescence Intensity comparison graph. Data represented as mean ±SD, *represents p < 0.05.



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hydrostatic pressure method, and implanted in rabbit cornea bydeep anterior lamellar keratoplasty. They could demonstrate re-epithelialization and satisfactory cellular infiltration after 3months. But main limitation of that study was initial cornealhaze and edema due to damaged or disordered ECM ofdecellularized matrics.58 Our study was able to resolve these

limitations, as our optimized protocol of decellularization ofcornea by perfusion bioreactor caused minimal disruption ofECM, and induced cell death by apoptosis, rather than necrosis,which caused suppression of the immune reactions. Interest-ingly, clinically and optically transparent host corneasconfirmed proof of concept that lamellar stromal transplants

Figure 6. Immunofluorescence analysis of CD63 staining of THP-1 monocyte on (A−C) NC, (G−I) DC, (J−L) DC+CS, and PMA-stimulatedmacrophage on (D−F) NC and (M−O) DC+CS cultured for 72 h. DAPI, CD63/AF555, and merged positive channel has been represented in 20×magnification. (P) Fluorescence Intensity comparison graph. Data represented as mean ± SD, *represents p < 0.05.



I

were well accepted by the host stromal bed. Our study alsogenerated valuable insights about modalities of macrophagematuration, and minimization of the innate immune responsewith respect to chemical modification of decellularized matrices.Moreover cross-linking of the decellularized cornea with CScontributed toward faster graft integration, cellular infiltrationand reduced the chances of cross species reaction.These interesting findings opened up many new directions.

First, this study can be further extended to the full thickness

stromal substitute in corneal transplantation among non-primates i.e. from goat to rabbit. But in order to extend theseinsights on the use of xenografts from nonprimates to primates,the absence of α-Gal epitope in the xenografts must be ensuredbefore implantation in primates. α-Gal (Galα1−3Galβ1−4GlcNAc-R) epitope present in the cell surfaces of thenonprimates generates immune response in humans due tothe presence of natural α-gal antibody comprising 1−3% of thetotal circulating immunoglobulin. These circulating antibodies

Figure 7. (A) Immunofluorescence analysis of LAMP-2 staining of THP-1 monocyte on (a−c) NC, (g−i) DC; (j−l) DC+CS, and PMA stimulatedmacrophage on (d−f) NC and (m−o) DC+CS cultured for 72 h. DAPI, LAMP-2/AF555, and merged positive channel has been represented in 20×magnification. (p) Fluorescence Intensity comparison graph. (B) Schematic representation of macrophage activation on corneal matrices. Datarepresented as mean ± SD, *represents p < 0.05.



J

can activate the complement and humoral immune system.Second, the sign of polarization in THP-1 derived macrophageare generally studied by means of expression of selectedtranscription factors or cytokines induced by different strong(PMA), followed by weak stimuli (LPS, IFN-γ) to concludeabout discrete differentiation state to either M1 (pro-inflammatory) or M2 phenotypes (anti-inflammatory).Although we could generate valuable insights about macro-phage maturation, but precise mechanism of decellularizedECM mediated polarization of macrophage is still not clear.Identification of the intermediate polarization stage (M0, M1a,M1b and M2a, M2b) needs to be done, to develop further

immunomodulatory constructs. Before moving to humanclinical trial,59 detailed sequential surface markers analysis isneeded to identify the proper subtype to predict clinicaloutcome more accurately. Different activation strategy can beadopted in near future to modulate the polarization ofmacrophage60 in a more favorable way to control the immuneresponse mediated by decellularized scaffolds, which can beextended to other tissues as well.

■ CONCLUSION

The present study demonstrates the potential use of a xenograftcorneal transplantation model by implanting decellularized

Figure 8. (A) Perfusion bioreactor containing cornea for decellularization (B) Peristaltic pump and the set up of the bioreactor, (C) H&E images ofthe whole rabbit cornea 3 months after implantation of native goat corneal stroma (NC) in the stromal pocket. Black arrows indicate the differentlayers starting from the (1) outer epithelium, (2) Bowman’s layer, (3) keratocyte nuclei present in the stroma, (4) stromal layer, (5) Descemet’smembrane, (6) endothelium layer, (D) magnified view of C, (E) implantation of the corneal matrix in the rabbit’s stromal pocket.



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corneas from goat in to the rabbit stromal region, which mayoffer potential to serve as an alternative to human cadaveric

corneas. The immune privilege nature of native cornea hasallowed us to examine the possibility of moderate immune

Figure 9. H&E images of the rabbit cornea 3 months after implantation of decellularized cornea (DC) (A) Intact (a1) Bowman’s and (a2)Descemet’s membrane; (a3) occasional gaps mostly present in (a4) the subepithelial region. (B) Separation of stroma lamella; (b1) magnified viewof B showing (b2) interlamellar space; (C) (c1) extensive cellular infiltration in stroma; (c2) magnified view of C, (c3) nuclei of rabbit keratocytes;(D)magnified view of the stratified epithelial region; (E) focal gaps in subepithelial region.

Table 1. Histological Examination

grouprabbitno.

lefteye right eye

cellularinfiltration vascularization inflammation

loss of nuclei instromal cells

stromal cellseparation

1. decellularized corneas R1 test control 2+ 3+ 1+ 1+ 2+R2 test control 1+ 2+ 0 focal 1+R3 test control 2+ 2+ 0 1+ 1+

2. decellularized corneas cross-linked withchondroitin sulfate

R1 test control 1+ focal 0 focal 0R2 test control 1+ 1+ 0 0 0R3 test control 2+ focal 0 focal focal

3. native corneas R1 test control 1+ 0 0 0 0R2 test control focal 0 focal 0 0R3 test control 1+ 0 0 0 0



L


response mediated by the decellularized cornea. Compared tonative goat corneal stroma, decellularized cornea exhibitedhigher immune response. Further cross-linking it with oxidizedchondroitin sulfate resulted in resolving immune responsecompared to the decellularized corneas. Detailed macrophagematuration mechanism has been studied by luminal acid-ification, endocytosis, lysosomal and endosomal content ondifferent experimental groups. In vivo study further validatedour previous in vitro findings. Apart from the potential of thexenograft for corneal reconstruction, this decellularized matrixscaffold can also be used as an in vitro model system inreproducing new understanding about different therapeuticcondition and corneal regeneration.

■ ASSOCIATED CONTENT*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acsbiomater-ials.8b00251.

Total GAG and DNA content of NC and DC, covalentcross-linking of collagen type I with chondroitin sulfate,tridimensional shape analysis of monocytes, and macro-phages cultured in the designated corneal matrices(PDF)Video S1, surgery video (ZIP)

■ AUTHOR INFORMATIONCorresponding Author*E-mail: [email protected].

Figure 10. H&E images of the rabbit cornea 3 months after implantation of cross-linked cornea (DC+CS). (A) Nominal/focal separation of thelamella; (a1) magnified view of A, separated lamella; (B) lamellar organization of stroma (b1); (b1) magnified view to show orientation ofkeratocytes along with ECM; (C) seamless blending of the graft in the stromal pocket; (c1) magnified view of C; (D) cellular infiltration in theimplanted graft region (d1); (d1) magnified view of D, orientation of cells and fibrous ECM.



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http://pubs.acs.org

http://pubs.acs.org/doi/abs/10.1021/acsbiomaterials.8b00251

http://pubs.acs.org/doi/abs/10.1021/acsbiomaterials.8b00251


http://pubs.acs.org/doi/suppl/10.1021/acsbiomaterials.8b00251/suppl_file/ab8b00251_si_002.zip

mailto:[email protected]


ORCIDSourabh Ghosh: 0000-0002-1091-9614Author Contributions†J.C. and S.R. contributed equally to this work

NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTS

This study was supported by IIT Delhi’s intramural fundingHigh Impact project, Department of Biotechnology (BT/MB/INDO-US/VR/09/2013 Dt. 31/10/17). We also acknowledgehelp of AIIMS Neurosurgery-DBT animal surgical facility.

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http://dx.doi.org/10.1186/s12885-015-1546-9

http://dx.doi.org/10.3389/fimmu.2017.01097

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