Modulation of Macrophage Phenotype, Maturation, and Graft

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Modulation of macrophage phenotype, maturation and graft integration through chondroitin sulfate conjugation to decellularized cornea Juhi 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 ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00251 • Publication Date (Web): 01 May 2018 Downloaded from http://pubs.acs.org on May 4, 2018

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ACS Biomaterials Science & Engineering

Modulation of macrophage phenotype, maturation and graft integration through chondroitin sulfate crosslinking to decellularized cornea Juhi Chakraborty1a, Subhadeep Roy1a, Sumit Muraba, Raghav Ravanib, Kulwinder Kaura, Saranya Devib, Divya Singhb, Shubhangini Sharmac, Sujata Mohantyc, Amit Kumar Dindad, Radhika Tandonb, Sourabh Ghosha a

Regenerative Engineering Laboratory, Department of Textile Technology, Indian Institute of Technology Delhi, India

b

Dr Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, India c d

1

Stem Cell Facility, All India Institute of Medical Sciences, India

Department of Pathology, All India Institute of Medical Sciences, New Delhi, India

Contributed equally

Corresponding Author Department of Textile Technology, IIT Delhi, New Delhi, India *E-mail: [email protected]

Abstract Decellularized corneas obtained from other species have gained intense popularity in the field of tissue engineering due to its role to serve as an alternative to the limited availability of high quality donor tissues. However the decellularized cornea is found to evoke an immune response inspite of the removal of the cellular contents and antigens due to the distortion of the collagen fibrils that exposes certain antigenic sites, which often lead to graft rejection. Therefore, in this 1

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study we tested the hypothesis that crosslinking the decellularized corneas with Chondroitin Sulfate may help in restoring the distorted conformationation changes of fibrous matrix and thus help in reducing the occurrence of graft rejection. Crosslinking of the decellularized cornea with oxidized Chondroitin Sulfate was validated by ATR-FTIR analysis. In vitro immune response study by involving healthy monocytes and differentiated macrophage with their surface marker analysis by pHrodo red, Lysotracker red, ER tracker and CD63, LAMP-2 antibodies confirmed that the crosslinked decellularized matrices elicited least immune response compared to the decellularized ones. We implanted three sets of corneal scaffolds obtained from goat i.e. native, decellularized and decellularized corneas conjugated with Chondroitin Sulfate 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, affirming proper graft integration. Hence we conclude that the Chondroitin Sulfate crosslinked decellularized corneal matrix may serve as an efficient alternative to the allograft and human cadaveric corneas.

Introduction The cornea is a key component in the optical path of the eye and also serves as a transparent physical barrier to the outer environment. Corneal diseases are one of the major causes of blindness that affects around 4 million people worldwide.1 Globally, there are relatively 1.5 million cases of corneal blindness investigated, while in India itself approximated 6.8 million people suffer from vision loss at least in one eye due to corneal diseases; which rises by 25,000-30,000 cases every year.2 Inspite of such an alarming situation estimated only 120,000 corneal transplants are undertaken annually.3 Presently, keratoprostheses and transplantation of cornea from deceased are primarily used for visual recovery.4 However these strategies often lead to glaucoma, infection, calcification, retinal detachment, corneal melting and prosthesis extrusion.5 The constrained availability of high quality donor tissue in many countries, standard therapy of immunosuppressive steroids after grafting and the rapid graft rejection in some patients have led to the evolution of tissue engineered corneas as a potential substitute to traditional corneal grafts. 2


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In attempt to fabricate tissue engineered cornea, corneal cells have been cultured into wide varieties of natural or synthetic polymeric scaffolds for e.g. recombinant human collagen hydrogel and recombinant human collagen 2-methacryloylxyethyl phosphorylcholine6, silk fibroin7,8, gelatin hydrogel9, gelatin-polyHEMA10 etc. Invariably it is impossible to replicate the precise anatomical microstructure and extracellular matrix (ECM) composition of the cornea. Presence of random pores in the polymeric scaffolds makes it difficult to control cellular orientation, achieve adequate strength and transparency - which are the major requirements in generating a functional cornea.11 Corneal transparency is governed by the orderly arrangement of collagen fibers in the stroma. The collagen type I and collagen type V fibers in the stroma, which are usually 25-30 nm in diameter, are organized in a parallel fashion with a regular spacing of 30 nm between each fibril. This precisely regular spacing is controlled by the presence of the proteoglycans around the collagen fibrils. This orderly array of fibers along with the proteins expressed by the keratocyte cells are known to play a significant role in corneal transparency.12 Degradation of the implanted biomaterials (crosslinked recombinant human collagen), haze at the interface between the host stroma and the graft, delayed epithelial closure caused postsurgical astigmatism, and highest corrected visual acuity of 0.4 could be achieved in six out of ten patients at 24 months.13 A four year long, phase-1 human clinical trial was conducted to replicate the corneal structure using cross-linked recombinant human Collagen type III and 2methacryloyloxyethyl phosphorylcholine, a synthetic phospholipid, as a corneal substitute in patients who were at high risk of graft failure. This strategy was able to restore vision in two out of three patients.14 Therefore the requirement of a more suitable biomaterial that can be colonized by host corneal cells easily, integrate with surrounding tissue, restore the corneal transparency is a prerequisite in fabrication of a functional cornea. As the polymeric scaffolds fail to replicate the native corneal ECM chemistry and architecture, corneas from other species such as pig, rabbit, sheep may serve as an alternative to human corneal tissue.15 The corneal allograft transplantation is expected to be successful due to the immune privileged nature of the corneal tissue.16 The reason for this immune privilegeness can be attributed due to the absence of blood vessels, lymphatic system in the cornea, the existence of immunomodulatory factors in the aqueous humor, and the blood/eye barrier. As a result the time taken between the corneal transplantation and antigen recognition gets extended, 3



thus arresting the entrance of the effector cells.17 In addition, low levels of MHC class I molecule expression and no expression of MHC 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 about the mechanism of immune privilege as corneal transplantation occasionally end up with inflammation and corneal allograft rejection.19 Decellularization of the corneas from other species (xenografts), from which the cells and antigen molecules are eliminated to decrease the host immune reaction, have gained increasing interest and are suggested to serve as a promising alternative to human cadaveric corneas. Lately, several groups have demonstrated decellularization of cornea by using a plethora of methods, including chemical means by using Tris buffer, ethylene diamine tetraacetic acid and sodium dodecyl sulphate20, by using enzymes such as nucleases, phospholipase A2, trypsin etc21,22 and physical methods such as freeze drying or high hydrostatic pressure.23 There is a strict requirement that the process of decellularization should absolutely remove all cellular components/material including the lipid membranes and antigen molecules within the tissue and membrane surface, solubilize the cytoplasmic and nuclear cellular molecules, DNA fragments, while simultaneously removing the cellular debris from the tissue, to reduce any possible host rejection or immunological reaction. At the same time the decellularization protocol must retain the structural, functional properties and integrity of the ECM.24 Preservation of pseudohexagonal lattice structure of collagen bundles is a crucial step while designing a protocol for decellularization, to ensure transparency. In addition, the decellularized corneal matrix should be efficient in sustaining the adhesion and proliferation of the corneal cells and therefore should act as a matrix resembling the native cornea.25,26 In our previous study, we compared various chemical and physical

methods of decellularization, where we concluded that the corneal

decellularization using a perfusion bioreactor with 0.1% Triton-X 100 (non-ionic detergent, toctylphenoxy polyethoxyethanol) resulted in an efficient removal of cellular and nuclear material.27 Unique finding of that study was, the controlled unidirectional flow of detergent at the rate of at 10 ml min-1 directly through the cornea persuaded cells to undergo apoptosis (but not necrosis) and assisted in efficient removal of cells, causing improved decellularization efficiency, while retaining the ECM ultrastructure. This may offer crucial implication for inflammatory response post transplantation in two ways. Firstly, decellularization of a tissue 4


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through apoptotic pathways is a preferred mode to induce cell death compared to necrosis trajectory, as this would trigger phagocytosis by immune cells, and suppress the immune reactions. But necrosis induced impaired membrane integrity may result in inflammation and graft rejection27. Secondly, in a follow up study28 we elucidated that although the disturbance to the ECM ultrastructure can be minimized by following this optimized slow perfusion-based decellularization strategy, excessive hydration and disruption of secondary conformation of the collagen stromal fibers cannot be entirely evaded. ATR-FTIR and Raman spectroscopic analysis showed that the α-helix to β-sheet ratios decreased significantly after decellularization by Triton X-100 treatment, along with increase in random coils and β-strands content. This insight generates a concern about the exposure of certain antigenic sites during the process of decellularization, which are otherwise buried within the triple helical structure. These two important aspects are often ignored in development of decellularized matrix for organ regeneration. At the same time, partial alteration in conjugation between collagen and decorin and keratan sulfate was noticed. Osmoregulatory agents such as glycerol, dextran could retrieve the native conformation of collagen fibrils present in the stromal layer, as well as facilitated reattachment of decorin or keratan sulphate molecules to the collagen molecules but they were insufficient in regaining the native structural properties of the cornea.29 Decellularization induced modulation in collagen conformation was further corroborated by Hwang et al30, through specific binding of a collagen hybridizing peptide with denatured collagen with altered conformation. It would be interesting to investigate how denatured collagen fibrils with modulated conformation at the decellularized corneal matrix may trigger innate immune response. At the same time, strategies to conjugate proteoglycans with collagen fibrils of decellularized ECM may help in restoring chemical composition of the stroma, while imparting specific biological functionality.31 A major component of decorin is the glycosaminoglycan (GAG) chain, consists of chondroitin sulfate, dermatan sulfate or their copolymer. Chondroitin Sulfate (CS) is a sulfated GAG made up of a chain of alternating glucoronic and N-acetyl galactosamine sugars.28 Crosslinking CS with N-hydroxysuccinimide (NHS) group resulted in an increase in the diameter and density of the collagen fibers and also improved the mechanical strength of the cornea in Keratoconus rabbit models.32 In addition, CS was known to play significant role in wound healing.28 CS is reported to play a crucial role in reducing immune 5



response32 and at the same time known to increase mechanical strength.33 Inflammatory responses in many tissues are tightly regulated by activation of NF-kB transcription factors. CS exerts anti-inflammatory activity by inhibiting the nuclear translocation of nuclear factor κ-lightchain-enhancer (NF-κB) of activated immune cells and have a stabilization effect on the DNA binding domain (NF-kBp65) by regulating its deacetylation34. Since it is an essential component of the ECM of many tissues including the cornea, we hypothesized that CS conjugation with decellularized corneal stromal ECM would result in enhanced graft integration, reduce foreign body reaction or innate immune response and tissue inflammation. Therefore, in the present study we attempted to address two questions. Firstly, whether native or decellularized corneas would inflict immune response when implanted from one species (goat) to other (rabbit). Our second aim was to find how crosslinking the decellularized corneas with CS would modulate the immune response, both in vitro and in vivo. Thus, we aimed to reduce the immune response of the decellularized cornea by crosslinking it with CS that would result in increasing its biomechanical strength by replenishing the proteoglycan loss and recovering its biochemical composition. To the best of our knowledge this is the first study on the use of CS for the crosslinking of decellularized goat corneas as an alternative method for generating corneal substitute for transplantation. We tested the potential of our decellularized crosslinked corneal constructs from goat as a corneal graft for transplantation into rabbit corneal stroma and validated this both in vitro and in vivo by studying the comparative effect of immune response generation using three sets of corneal scaffold (native cornea- NC, decellularized cornea- DC and decellularized cornea crosslinked to oxidized chondroitin sulfate- DC+CS). Materials and Methods Cornea Isolation Cadaveric goat eyes were collected from All India Institute of Medical Sciences, New Delhi, with prior approval from the institute ethical committee. Corneas form the goat ocular globe were excised under sterile conditions and washed five times in PBS containing 100 U/ml penicillin, 50 µg/ml gentamycin (Himedia, India), 100 µg/ml amphotericin B (Himedia, India) and streptomycin (Lonza, USA).

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Decellularization The dissected tissue was decellularized using a perfusion bioreactor (kindly provided by Prof. Ivan Martin, Basel, Switzerland)35, as described elsewhere27. Briefly, Triton-X 100 (0.5 % in PBS) was perfused directly through the corneal tissue using a peristaltic pump (KD Scientific, USA) unidirectionally at a constant flow rate of 50 µl/min for 48 h. Thereafter, the decellularized tissue was rinsed through the same bioreactor by perfusing antibiotic mixture at a flow rate of 10 µl/min for 48 h. Crosslinking with Chondroitin Sulphate (CS) CS (from bovine trachea, MW 105 Da., Merck, Catalogue No. 6A2942) was oxidized using periodate salt. Briefly, 616 mg of NaIO4 and 600 mg of CS were dissolved in 10 ml of deionized water at dark. The reaction was continued for 1 h, with vigorous stirring at 40°C. The reaction product was purified by filtration with Sephadex G-25 (Sigma) size-exclusion chromatography36. In next step, decellularized corneas were first rinsed for 1 h with sodium acetate solution (10% w/v), thoroughly washed with deionized water and subjected to crosslinking with oxidized CS (6% w/v in distilled water). Finally crosslinked decellularized corneas were thoroughly washed in deionized distilled water to ensure that there was no unbound CS left. Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) ATR-FTIR spectrum of the freeze dried samples (NC, DC, DC+CS) was obtained using an Alpha-P spectroscope (Bruker, USA). An infrared spectrum was acquired in transmittance mode in the spectral region of 400–4000 cm−1 and a spectral resolution of 4 cm−1, and 240 scans were taken for each sample. Origin 8.5 was used for obtaining the peaks in the given spectral region. In vitro Immune Response Study Cell culture Human monocyte cell line THP-1 (derived from acute monocytic leukaemia) was maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), penicillin-streptomycin, gentamicin and 2 mM sodium pyruvate. THP-1 monocytes were differentiated into macrophages by treating with phorbol-12-miristate-13-acetate (PMA) at a concentration of 20 ng/ml, in 6 well 7



microplates at a concentration of 5.6×105 cells per well in complete RPMI 1640 media.37 After 72 hr, differentiated cells were washed with PBS for three times and then seeded with the fresh culture medium with different groups of corneal scaffold (NC, DC+CS). Freshly cultured monocytes were also incubated 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 and dehydration in graded alcohol series and air drying. The samples were then sputter coated with gold (up to 15–20 nm thickness) using an EMITECH K550X (UK) sputter coater set at 25 mA for 240 s. The coated samples were imaged at varied magnifications using a JEOL 5610LV (JEOL; Japan) SEM at an accelerating voltage of 5 kV. Initiation of fluid phase uptake or bulk phase endocytosis in THP-1 cells After incubation of monocytes and differentiated macrophages with different corneal scaffolds, 50 ng/ml concentration of pHrodo Red Dextran stain (Thermo Fischer Scientific, India) was added. After incubating the cells for a period of 1 hr, the constructs were rinsed twice with 10 mM PBS and were fixed for 15 min at room temperature in dark with BD cytofix/cytoperm assay kit. Cornea scaffolds were treated with background suppressor for respective blue and red channel to decrease signal-to-noise ratio and non-specific labeling and air dried and mounted in fluorescent mounting medium Prolong Gold antifade reagent with DAPI in a one end frosted glass slide and their fluorescence was observed and recorded on inverted confocal laser scanning 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). The corrected total cell fluorescence was calculated as a measure of fluorescence intensity using ImageJ (NIH). Lysosomal and Endosomal trafficking in THP-1 cells THP-1 cells were seeded on corneal matrices for 72 hr 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, Molecular Probes, USA) at 37°C for 2 hr. Subsequently samples were washed twice and fixed and permeabilized (BD Cytofix/ Cytoperm kit, USA) for 20 min at room temperature, washed with PBS and were treated with Signal Enhancer for the period of 45 min, stained with 8


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DAPI (Invitrogen, Italy) and mounted with Prolong Gold antifade mounting medium. Samples were treated with background suppressor for respective blue and red channel to decrease signalto-noise ratio and non-specific labeling. The samples were viewed by confocal scanning microscopy 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 ER tracker positive region by using an oil immersion 20X objective lens. Frame averaging were done with 3-4 scan to reduce noise. The corrected total cell fluorescence was calculated as a measure of fluorescence 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 FX signal enhancer for duration of 45 min. Cell endogenous peroxidase activity has been quenched by adding 3% H2O2 into the corneal scaffold. Then the samples were incubated with 100-150 µl blocking buffer (10% goat serum and 3% BSA) for a period of 2 hr in room temperature and processed with primary antibody against CD63 (Origene, TA802751), LAMP-2 (Origene, TA336932). All primary antibodies were diluted with Blocker BSA (Thermo Fisher, 554657) in a dilution of 1:500 and applied 500 µl for each samples for overnight at 4ºC. After the primary labeling, the cells were washed three times with wash buffer and incubated with poly-HRP-conjugated secondary antibody for 2 hr at room temperature. 100 µl of the tyramide working solution (Thermo Fisher, B40916) was added to each sample for a period of 2-10 min. Reaction was stopped by adding 100 µl of reaction stop reagent. Tyramide conjugation (Tyramide, H2O2, Reaction buffer) was performed to get a superior signal from poly-HRP conjugated secondary antibody with photostable bight Alexa Fluor 555. Corneal scaffolds were washed three times with PBS and mounted with Prolong Gold antifade mounting medium containing DAPI. Indirect immunofluorescence was examined using a confocal 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 were converted to 3D using the Leica LAS X 3D Visualization software (Leica Microsystems, Germany). The corrected total cell fluorescence was calculated as a measure of fluorescence intensity using ImageJ (NIH). 9



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 from the middle and punctured with a biopsy punch to take the circular corneal construct from the stroma. Adult New Zealand white rabbits were used for the experiments. 12 week old, two males and one female rabbits weighing 1.62-2.12 Kg were taken for each group (n=3). The rabbits were treated following the ARVO Statement on the Use of Animals in Ophthalmic and Vision Research. All animal experiments were approved by the institutional ethical and animal ethics 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 ketamine hydrochloride (35 mg/kg) (Trolkaa Pharmaceuticals Ltd., Dehradun, India). Left eye was implanted with the corneal implants in each rabbit. The recipient cornea was trephinated with a 4 mm biopsy punch to make the pocket. Decellularized corneas were trephinated using 3 mm biopsy punches to fit them into the recipient bed. The donor corneal implant was trephinated using a 4 mm biopsy punch and cut thereafter. The constructs were inserted into the corneal pocket and sutured with 10–0 nylon cardinal sutures (Video S1). Topical steroid- 1% w/v Prednisolone acetate (Sun Pharma Laboratories Ltd., Mumbai, India), antibiotic- Moxifloxacin (FDC Ltd. Aurangabad, India) and lubricant0.3% hypo-mellosebenzalkolium chloride (Sunways India Pvt. Ltd., Ahmedabad, India) were administered thrice daily for 3 weeks. After three months the rabbits were sacrificed by an overdose of sodium pentobarbital (200 mg/Kg). Both the eye balls of all the rabbits were excised out of the orbits and immersed in 4% paraformaldehyde solution for fixation. Microscopic grading of corneal haze Corneal grading system reported by Fantes et al38 was used for the grading of the corneal haze: Grade 0- cornea that was completely clear, Grade 0.5- cornea with a slight haze by slit lamp microscopic examination, Grade 1- haze which is not interfering with the visibility and noticeable fine details of the iris, Grade 2- slight obstruction of the details of the iris, Grade 3- a moderate obstruction of both the lens and the fine iris details, Grade 4- no visibility of the 10


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corneal details due to complete opacification of cornea. The grading of the corneal haze was done by three independent researchers 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 for histological examination. Five micron thick paraffin sections were taken for H&E staining. Subsequently five sections for each group were examined under a light microscope. The images were captured for further analysis (Leica DFC295, Germany) using Leica software application suite (LAS V3.8).A grading scale of 0-3 was used to determine cellular Infiltration, vascularization, inflammation, loss of nuclei in stromal cells and stromal cell separation. The grading system used is: (A) Cellular infiltration & Inflammation (Ten consecutive high power fields (HPF) (20 x) were assessed under microscope (E 600, Nikon Corporation, Japan). Grading: 0 = no cell/10 HPF, 1= 1-5 cells /10 HPF, 2 = 6 – 10/10 HPF, 3 = >10 cells /10 HPF. (B) The grading of vascularization was done in ten consecutive high power fields (20 x) in the area of hotspot (where the vessels were 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 power field (20 x) in the stromal region was evaluated and grading was done 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 ± standard deviation, with n representing the number of experiment repeated. Student’s t-test was carried out to establish statistical significance and probability at p < 0.05 was considered significant. Each experiment was conducted with n=3 and were repeated twice. Results and Discussion

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Characterization of decellularized cornea was done in terms of residual GAG and DNA content (Table S1), as well as absence of cell nuclei, as reported earlier27. Crosslinked corneal ultrastructure analysis by ATR-FTIR spectroscopy In order to understand the interaction between the corneal collagen type I and the oxidized chondroitin sulphate (CS), FTIR spectrum of the studied samples were obtained (Figure 1). After cross linking the DC with oxidized CS followed by washing steps, it was possible to identify characteristic bands of CS in the spectrum. Amide I is the most sensitive band for detecting changes in the corneal stromal collagen type I layer and its characteristic transmission band was found to be located in the range 1600-1700 cm-1.39 The Amide I peak which is centered at 1630 cm-1 corresponds to the C=O stretching vibration.40 This amide band was shifted to lower wave number i.e. 1623 cm-1 in case of DC+CS sample. The observed “red shift” was due to the chemical interaction between the COO- and SO3- of CS and C=O of collagen fibrils. An increase in the intensity of the peak at 1623 cm-1 is due to the overlapping of amide I and OH vibrations with maximum 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 presence of N-H plane band and the C-H stretch vibration41 and C-O stretching vibrations.42 Vibration at 1241 cm-1 was assigned to amide III vibration of cornea epithelial Collagen type I layer43. Band near 1350 cm-1 was assigned to the symmetric methyl bending vibrations of the acetate anion of CS. Band near 1070 is due to C-O-C stretching.44,45 No peak corresponding to structural changes of corneal collagen type I component was found i.e. in the range of 1610-1620 cm-1. Band near 3300 cm-1 was due to O-H vibrations in all the samples. The crosslinking time and density was optimized using a number of corneas and only the most optimized result has been reported. The steps involved and the mechanism of crosslinking has been explained in the experimental protocol (Figure S1). Hence, on the basis of FTIR analysis we concluded that the oxidized CS binds with C=O of the corneal collagen type I through COO- and SO3- linkage. This crosslinking may help collagen type I layer to regain its stability after the decellularization procedure and thus maintaining lamellar orientation akin to native cornea.46,47 Covalent tethering of CS with collagen fibrils were reported to result in increased fibril density and oriented lamellae when compared to fibrils in only DC.48 CS conjugation also reported to preserve the

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regular collagen fibrillary dimensions compared to the destructive corneal collagen architecture in DC.49 SEM Analysis SEM analysis was done to determine the morphology and surface architecture of the THP-1 monocytes 72 hrs after seeding on corneal scaffold. The monocytes (without PMA treatment) grown on two groups of corneal scaffold (NC and DC+CS) displayed spherical morphology with nominal ruffles and blebs noticeable on their surfaces. However the THP-1 monocytes culture on the DC scaffolds differentiated into macrophages, without any chemical activation, which was evident due to increase in their cell size (103.77 µm2), feret’s 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

Modulation of Macrophage Phenotype, Maturation, and Graft

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