Nanotechnology and Regenerative Medicine 229 Ocular ... - ARVO

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Nanotechnology and Regenerative Medicine229 Ocular Nanotherapy, Nanoparticles, and Gene TherapyMonday, May 06, 2013 8:30 AM-10:15 AMExhibit Hall Poster SessionProgram #/Board # Range: 1383-1410/B0001-B0028Organizing Section: Nanotechnology and Regenerative MedicineProgram Number: 1383 Poster Board Number: B0001Presentation Time: 8:30 AM - 10:15 AMUse of Polymeric Scaffolds as Extracellular Matrix Substitutesfor Conjunctival Epithelial RepairThomas Storr-Paulsen 1, 3 , Matthew Fullana 2 , Annie L. Wang 1 ,Dimitrios Karamichos 1 , Tor P. Utheim 1, 4 , Marie A. Shatos 1 , JesperHjortdal 3 , Gary E. Wnek 2 , Darlene A. Dartt 1 . 1 Schepens EyeResearch Institute, Massachusetts Eye and Ear, Harvard MedicalSchool, Boston, MA; 2 Department of Macromolecular Science andEngineering, Case Western Reserve University, Cleveland, OH;3 Department of Ophthalmology, Aarhus University Hospital NBG,Aarhus, Denmark; 4 Department of Medical Biochemistry, OsloUniversity Hospital, Oslo, Norway.Purpose: The aim of this study is to investigate the use of artificialscaffolds to replace the native extracellular matrix (ECM) and use forthe repair of wounded conjunctiva. We hypothesize that a polymericscaffold, that approximates the mechanical properties of the nativeconjunctiva, will induce optimum growth and cellular viability ofconjunctival goblet and stratified squamous epithelial cells.Methods: Using electrospinning techniques, we fabricated porouspolymeric scaffolds made of poly(caprolactone) (PCL), poly(vinylalcohol) (PVA) and crosslinked poly(acrylic acid) (PAA).Polyethylene terephthalate (PET) scaffolds in tissue culture insertswere purchased and used for comparison. Explants from cadaverichuman conjunctival tissue were placed on each scaffold and culturedin 10% FBS supplemented RPMI-1640 medium for 12 to 15 days.After fixation and DAPI staining, outgrowth area relative to explantsize was calculated. A live-dead assay was performed, and thepercentage of viable cells was calculated after confocal laserscanning microscopy. Scaffolds with cultured cells were processedfor immunocytochemistry (ICC) and the expression of markers forstratified squamous epithelial cells (cytokeratin 4) and goblet cells(cytokeratin 7) were visualized by confocal laser scanningmicroscopy.Results: The percentage of viable cells was 89.9±5.6% (n=5) forPAA, 3.6±2.2% (n=5) for PCL, 0% for PVA (n=3) and 92.7%±5.4%for PET (n=3). The difference between PAA and PET was nonsignificant(p=0.938), whereas both PCL and PVA was significantlydifferent from PET (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Nanotechnology and Regenerative Medicineminutes, 192.3 ± 141.9 and 7.8 ± 4.4 after 30 minutes, 51.7 ± 37.5and 3.4 ± 2.1 after 45 minutes, 46.3 ± 57.4 and 4.3 ± 6.6 after 60minutes and 7.4 ± 5.4 and 2.2 ± 1.2 after 120 minutes afteradministration, respectively. The difference between DexNP andMaxidex® was highly significant at all time points (p < 0.001).Conclusions: There was a highly significant difference inconcentration of dexamethasone in the tear fluid at all time pointsbetween DexNP and Maxidex®. The concentration was up to 140fold higher in the DexNP eyes than the Maxidex® eyes. The resultsindicate a greater mucoadhesion of DexNP which can explain themore long lasting effect of the cyclodextrin nanoparticle drugdelivery platform.Commercial Relationships: Gauti Johannesson, Allergan (C),Alcon (C); Maria D. Moya-Ortega, Oculis ehf. (E); Gudrun M.Asgrimsdottir, Oculis ehf. (E); Thorsteinn Loftsson, Oculis ehf (P);Einar Stefánsson, Oxymap ehf (P), Oxymap ehf (I), Oculis ehf (P),Oculis ehf (I), Risk ehf (I), Acta Ophthalmologica (E)Support: Unrestricted grants from Sjonverndarsjodur and theSwedish Society of MedicineProgram Number: 1386 Poster Board Number: B0004Presentation Time: 8:30 AM - 10:15 AMEngineering a biocompatible cell carrier with nanofeaturedtopography for retinal pigment epithelium transplantationZengping Liu 1 , Na Yu 2 , Frank G. Holz 1 , Fang Yang 2 , Boris V.Stanzel 1 . 1 Ophthalmology, University of Bonn, Bonn, Germany;2 Department of Biomaterials, Radboud University Nijmegen MedicalCenter, Nijmegen, Netherlands.Purpose: An optimal carrier is crucial for retinal pigment epithelium(RPE) tissue engineering. We investigated the influence of fibrillarvs. smooth topography on RPE behavior and subretinalbiocompatibility using biodegradable & biostable polymers.Methods: Poly L-lactide/ε-caprolactone (PLCL) or poly ethyleneterephthalate (PETP) fibrillar and smooth substrates were prepared byelectrospinning or a heating-press method, respectively. Topographywas characterized by scanning electron microscopy. Fetal hRPEgrowth curves were established from substrates seeded at 1×10E4cells/cm2, while for differentiation cells were seeded at 6×10E4cells/cm2. Morphology was monitored by phase contrast microscopy.Cultures were stained for tight junctions (ZO-1) at 6 weeks.Commercial membranes (Transwell®, Corning) coated with 200nmdiameter PLCL or PETP fibers were implanted into the rabbitsubretinal space. Repetitive in vivo images of the implants by SD-OCT and fundus photography were obtained after 4, 7 and 14days,followed by perfusion-fixed histology.Results: Uniform PLCL fibers were achieved in 209±33, 568±187 &1132±299nm diameter ranges, while PETP fibers were in widerranges (175±86 & 993±596nm). RPE attached on all substrates atcomparable densities (8958±2495 cells/cm2) at 24 hours. Cellamount on 200nm PLCL was 13.3% higher than on smooth, whileincreasing fiber diameters decreased cell counts at day 5. Cells on200nm PETP showed decreased cell amount (33.6%) compared tosmooth. In long term cultures, RPE monolayers partially/entirelydetached from PLCL (13/15) and PETP (9/15) smooth films. Celllayers were maintained on all fibrillar scaffolds. 200nm PLCL fibersinduced superior RPE pigmentation and uniformly hexagonal ZO-1staining compared to other PLCL fibrillar or smooth surfaces.Intraoperative handling of all implants was safe. The retina overlyingPET/PETP or PLCL subretinal implants appeared attached andtransparent on ophthalmoscopy, with largely preserved andcontinuous layering on SD-OCT. On histology, outer nuclear layerthickness was reduced by half and photoreceptors inner segmentswere preserved in some regions above both implants.Conclusions: RPE cells showed material and topography-dependedproliferation behavior. Scaffolds created by 200nm fiber diametertopography induced superior RPE differentiation. Both substrateswere well tolerated in subretinal space.Commercial Relationships: Zengping Liu, None; Na Yu, None;Frank G. Holz, Acucela (C), Allergan (C), Genentech (F),Heidelberg Engineering (F), Zeiss (F), Novartis (F), Novartis (C),Optos (F), Merz (C), Bayer (F), Bayer (C), Boehringer Ingelheim(C); Fang Yang, None; Boris V. Stanzel, Geuder AG (F), GeuderAG (P)Support: State Scholarship Fund by China Scholarship Council2008627116 (China) to ZPL, Gerok/BONFOR O-137.0015 to bvs,Dr. Ruediger Foundation to bvsProgram Number: 1387 Poster Board Number: B0005Presentation Time: 8:30 AM - 10:15 AMProliferation of porcine conjunctival fibroblasts in fibrin-basedscaffolds using alamar blue assayAna Fernández 1 , Jennifer Ramos 1 , Marta López 1 , Patricia Pérez 1 ,Elizabeth Santín 1 , Leire Llorente 1 , Yolanda Diebold 2 , F. JavierIglesias 1 . 1 Human Tissue Bank, San Francisco Clinic Foundation,León, Spain; 2 Ocular Surface Group, IOBA-University of Valladolid,Valladolid, Spain.Purpose: Cell proliferation rate is a common biocompatibilityparameter analyzed in tissue engineering when a new biomaterial istested. Our aim was to analyze the suitability of the AlamarBlueproliferation assay (AB) to measure cell proliferation intridimensional (3D) constructs using porcine conjunctival fibroblastsgrown in 3D fibrin-based scaffolds.Methods: Porcine eyes, obtained from a local slaughterhouse, wereenzimatically digested (collagenase type I solution for 24 h) in orderto obtain conjunctival fibroblast. Fibroblasts were expanded untilfifth passage in standard cell culture conditions. To determineoptimum cell density to be used as our standard in the AB assay,eight cell seeding densities, ranging from 1x104cells/ml to1,5x106cells/ml, were assayed in monolayer culture (n=5).Percentage reduction of AB by fibroblasts was calculated usingfluorescence measurements at 530nm excitation and 590 emissionwavelengths after 4 h incubation (AB assay recommendation).Measurements for each cell density were obtained as standardstraight lines. Scaffolds (n=20) were prepared from humancryoprecipitate or plasma at low (LC, 1,5mg/ml) or high (HC,3,5mg/ml) fibrinogen concentrations. The same 8 cell densities wereseeded in fibrin scaffolds and AB measured every h up to 30 hs. Atotal of 240 samples were analyzed.Results: Fluorescence signal obtained from fibroblast seededscaffoldsreached the standard straight line at different AB incubationtimes. Average incubation time for LC cryoprecipitate and LCplasma scaffolds was 26 hs, while HC cryoprecipitate and HC plasmascaffolds required 28 hs and 30 hs, respectively.Conclusions: Current knowledge about AB assay indicates that cellproliferation can be measured after 4 hour incubation regardless theused culture surface. Our study demonstrates that there is nodependence between type of culture surface and the incubation timerecommended by the AB standard protocol. Rather, the material usedfor cell culture and, in our case, fibrinogen concentration in itdetermines optimum measurement protocol. It is relevant to considerthis fact when biocompatibility of new materials is tested.Commercial Relationships: Ana Fernández, None; JenniferRamos, None; Marta López, None; Patricia Pérez, None;Elizabeth Santín, None; Leire Llorente, None; Yolanda Diebold,None; F. Javier Iglesias, NoneSupport: FEDER-CICYT Grant MAT2010-20452-CO3-01/03©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Nanotechnology and Regenerative MedicineProgram Number: 1388 Poster Board Number: B0006Presentation Time: 8:30 AM - 10:15 AMNovel pentablock copolymers as a nanotechnology platform forcontrolled ocular delivery of proteinsSulabh Patel, Gyan Mishra, Viral Tamboli, Ashim K. Mitra.Pharmaceutical Sciences, University of Missouri Kansas City,Kansas City, MO.Purpose: Current macromolecular delivery in the treatment of wet-AMD requires frequent intravitreal injections to maintain therapeuticlevels at retina/choroid. Frequent administrations can cause potentialcomplications like endophthalmitis, retinal detachment, retinalhemorrhage, and patient noncompliance. The objective of thisresearch is to synthesize and evaluate novel tailor-made pentablock(PB) copolymers for the controlled and non-invasive delivery ofmacromolecules in the treatment of posterior segment diseases.Methods: We have synthesized novel biodegradable PB copolymersby sequential ring opening polymerization. Different ratio and MWof each block (polyglycolide, polyethylene glycol, polylactide andpolycaprolactone) were selected for synthesis to optimize the releaseprofile of FITC-BSA, IgG and avastin from nanoparticles andthermosensitive gel. Nanoparticles were characterized for particlesize, poly dispersity, entrapment efficiency and drug loading. In vitrorelease studies of FITC-BSA, IgG and avastin from nanoparticlesalone and nanoparticles suspended in thermosensitive gel wereperformed in PBS (0.1M, pH 7.4) at 37 C. CD spectroscopy, ELISA,cell proliferation and cell invasion techniques were employed toevaluate conformation and binding affinity of released IgG andavastin. In vitro cell viability (MTS and LDH assay) andbiocompatibility studies were performed on various ocular (ARPE19, SIRC and HCEC) and macrophage cell lines (RAW 264.7),respectively.Results: FITC-BSA and IgG loaded nanoparticles prepared from PBcolpolymers exhibited ~ 40% and ~ 25% of burst release within first2 days followed by sustained release up to 40 and 52 days,respectively. IgG loaded nanoparticles suspended in thermosensitivegel demonstrated continuous zero order release up to 62 days.Moreover, CD spectroscopy and ELISA showed retention ofconformation and binding affinity of released IgG. Avastin loadednanoparticles demonstrated similar patterns of burst release andsustained release as of IgG loaded nanoparticles. Furthermore, cellmigration and cell invasion assay confirmed retention of biologicalactivity of the avastin. PB copolymers exhibited excellentbiocompatibility with negligible toxicity.Conclusions: This approach can act as a platform for ocular deliveryof therapeutic macromolecules, which can minimize the side effectsassociated with frequent intravitreal injections.Commercial Relationships: Sulabh Patel, None; Gyan Mishra,None; Viral Tamboli, None; Ashim K. Mitra, NonePurpose: To demonstrate that magnification and image processing inretinal prosthesis with an external camera and processor can enable avisual acuity well beyond the limit set by theoretical resolution of theimplanted array.Methods: To date, more than 50 patients blinded by outer retinaldystrophies have received an Argus epi-retinal prosthesis (SecondSight, Sylmar, CA). The Argus II System, which is commerciallyavailable in the European Economic Area, is the only retinal implantapproved for commercial use in these patients anywhere in the world.In a retinal prosthesis, the retina is stimulated based on the light levelin the receptive field of the electrode, directly or (as in the case of theArgus II) via a video camera. As a result, the spatial resolution isnominally set by the number of electrodes and the distance betweenneighboring electrodes. To date the best nominal acuity achieved hasbeen 20/1260.In the current study, the video image acquired by a high resolutioncamera was processed before being wirelessly transmitted to theimplant. While the field-of-view of the cells in the retina covered bythe Argus II array is about 20 degrees diagonally, in the currentexperiment the subject was able to reduce or magnify the image in arange from 0.4x to 16x using a remote hand-held controller (LogitechR-400). In addition, image enhancement was done by extractingglobal features in the image.Visual acuity was measured using a grating visual acuity test inwhich square-wave gratings at four orientations were presented on acomputer monitor and subjects were required to report the orientationof the bars within the 5 second time limit. In addition, the subject wasasked to read short words from a notebook with 2.3cm letters.Results: Using a high magnification of 16x the subject’s visualacuity was measured at 1.0 logMAR (20/200). For the reading task,the subject chose to use 4x magnification and was able to readaccurately from a notebook at a distance of 30 cm.Conclusions: Variable magnification and other image processingstrategies can extend the functionality of a retinal prosthesis beyondthe spatial resolution set by the number and spacing of the electrodes.This may prove to be a unique advantage of a retinal prosthesis thatemploys an external camera and processor, such as the Argus II.Program Number: 1389 Poster Board Number: B0007Presentation Time: 8:30 AM - 10:15 AMAcuboost: Enhancing the maximum acuity of the Argus IIRetinal Prosthesis SystemJose A. Sahel 1, 2 , Saddek Mohand-Said 2, 1 , Paulo E. Stanga 4, 3 , AviCaspi 5 , Robert J. Greenberg 5 . 1 UMR-S 968, Institut de la Vision,Paris, France; 2 CHNO des Quinze-Vingts, INSERM-DHOS CIC 503,Paris, France; 3 Manchester Academic Health Science Centre andCentre for Ophthalmology and Vision Research, Institute of HumanDevelopment, University of Manchester, Manchester, UnitedKingdom; 4 Manchester Vision Regeneration (MVR) Lab, ManchesterRoyal Eye Hospital, Manchester, United Kingdom; 5 Second SightMedical Products, Sylmar, CA.Commercial Relationships: Jose A. Sahel, UPMC/Essilor (P),Second Sight (F); Saddek Mohand-Said, None; Paulo E. Stanga,OPTOS PLC (F), OPTOS PLC (C), OPTOS PLC (R), TOPCONCORP (F), TOPCON CORP (C), TOPCON CORP (R), SECONDSIGHT (F), SECOND SIGHT (R); Avi Caspi, Second Sight MedicalProducts, Inc. (C), Second Sight Medical Products, Inc. (P); RobertJ. Greenberg, Second Sight Medical Products, Inc. (I), Second SightMedical Products, Inc. (E), Second Sight Medical Products, Inc. (P),Second Sight Medical Products, Inc. (S)Clinical Trial: NCT00407602©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Nanotechnology and Regenerative MedicineProgram Number: 1390 Poster Board Number: B0008Presentation Time: 8:30 AM - 10:15 AMLab-on-a-tube for in situ ocular bioassays: continuousglucometry in tearsAndrew W. Browne 1 , Chunyan Li 2 . 1 Ophthalmology, University ofSouthern California Doheny Eye Institute, Los Angeles, CA;2 Cushing Neuromonitoring Laboratory, Feinstein Institute forMedical Research, Manhasset, NY.Purpose: Non-invasive monitoring is an expanding frontier forbiomedical assays. Integrated Bio-Micro-Electro-Mechanical-Systems (BioMEMS) have enabled miniaturization of clinical assaysinto integrated testing platforms for the point-of-care (POC).BioMEMS have the ability to push the envelope on blood tests at thePOC. Tear film glucometry and the methods for tear sampling mayobviate the need for painful finger sticks, and facilitate tighter bloodglucose control in diabetic patients. We propose to develop a lab-ona-tube(LOT) platform for continuously monitoring tear dynamics.Methods: Platinum nanoparticle-coated gold (Pt NP, working andcounter), and Iridium oxide (reference) microelectrodes werefabricated on 7.5 μm thick polyimide film. The Pt NP electrodes werecoated with Poly(ο-phenylenediamine) (PPD) to eliminateinterference. GOD-chitosan-glutaraldehyde solution was depositedfor a favorable glucose oxidase (GOD) microenvironment. Theflexible LOT may have sensors on either inner or outer surface.Artificial tear solution was prepared with 130 mM sodium chloride,10 mM dibasic sodium phosphate and 10 mM monobasic sodiumphosphate, lysozyme 1.9 mg/mL, albumin 0.2 mg/mL and mucin 0.15mg/mL in deionized water, at pH 7.4. Electrochemical glucosedetection was performed with a PalmSens® at 37°C.Results: Figure 1 shows the LOT. Incremental increases in glucoseconcentration were measured in artificial tear solution (Fig.2). Thesensor demonstrated a linear response range, sensitivity and detectionlimits of 0.01 - 2 mM, 35.8 nA/mM and 0.01 mM, respectively. Theglucose sensor sustained sensitivity for 7 days of continuous activitywith 95% original response retention.Conclusions: The LOT demonstrates high sensitivity for glucoseconcentrations characteristic of the tear film. With our previous datademonstrating stability of glucose sensors in artificial CSF over theperiod of months, our early platform for tear glucometry may providea means for long term ocular glucometry for months at a time.Ultimately, this tube architecture could find utility for glucometry insmart punctal stents or glaucoma drainage implants.Figure 2: Current output from glucose sensor exposed toincrementally increased glucose concentration in artificial tearsolution.Commercial Relationships: Andrew W. Browne, None; ChunyanLi, NoneProgram Number: 1391 Poster Board Number: B0009Presentation Time: 8:30 AM - 10:15 AMThe effect of retinoic acid to induce one of the perioculer neuralcrestMotokazu Tsujikawa 1 , Erika Kimura 1 , Yoshinori Oie 1 , TakeshiSoma 1 , Susumu Hara 1 , Ryuhei Hayashi 1 , Shin Hatou 2 , SatoruYoshida 2 , Shigeto Shimmura 2 , Kohji Nishida 2 . 1 Ophthalmology,Osaka University, Suita, Japan; 2 Ophthalmology, Keio University,Tokyo, Japan.Purpose: Corneal endothelium (CE) is developed from neural crest(NC) cells. NC emerges at the interface between neural and nonneuralectoderm and migrates extensively to form skeleton, cornea,teeth, thymus adrenal glands, and other tissue. To develop CE fromhuman induced pluripotent stem (iPS) cells, we developed a protocolto derive NC cells from human iPS cells. However, these NC cells donot express cranial and especially periocular NC markers. In thisresearch, we investigated the effect of retinoic acid to induce one ofthe perioculer NC markers, PITX2.Methods: iPS cells (253G1) were cultured for weeks by Sphereculture without serum, and NC cells were isolated by FACS usingp75NTR as a marker. These NC cells were seeded on dishes withretinoic acid in various concentrations. The cells were harvested andthe expression of PITX2 was quantified by RT-PCR method. Toobtain highest expression of PITX2, the concentration and theduration of RA application was modified. We also investigate NCmarkers, p75NTR and SOX10.Results: PITX2 expression level showed a peak 5 to 10 days afterapplication of RA. (

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Nanotechnology and Regenerative MedicineCommercial Relationships: Motokazu Tsujikawa, Shionogi & Co.(C), Daiichi Sankyo Co. (F), Daiichi Sankyo Co. (R), Santen Co. (R),AMO Co. (R); Erika Kimura, None; Yoshinori Oie, Santen (F),HOYA (F); Takeshi Soma, HOYA corporation (R), SantenPharmaceutical Co., Ltd (F), Otsuka Pharmaceutical Co., Ltd (F);Susumu Hara, None; Ryuhei Hayashi, None; Shin Hatou, None;Satoru Yoshida, None; Shigeto Shimmura, None; Kohji Nishida,Alcon (C), Alcon (F), HOYA (F), Senju (F), Pfizer (F), Santen (F),Osaka University (P)Support: Grant fromMinistry of Education,Culture,Sports,Science &Technology in Japan (To K. N, and M. T.)Program Number: 1392 Poster Board Number: B0010Presentation Time: 8:30 AM - 10:15 AMTissue Engineered Model of the Outer Neural Retina and RetinalPigment EpitheliumKarl E. Kador 1, 2 , Enrique Salero 1, 2 , Kristina R. Russano 1, 2 , Lung W.Lau 1, 2 , Jeffrey L. Goldberg 1, 2 . 1 Ophthalmology, Bascom Palmer EyeInstitute, Miami, FL; 2 Interdisciplinary Stem Cell Institute,University of Miami Miller School of Medicine, Miami, FL.Purpose: Retinal degenerative diseases (RD) that targetphotoreceptors (PR) or the adjacent retinal pigment epithelium (RPE)affect millions of people worldwide and is characterized by theprogressive loss of PR that are largely responsible for vision. Theetiology is attributed primarily to RPE cells that exist as a monolayerunderlying and supporting the neural retina. Effective treatment forRD have been widely investigated, however there is no in vitro modelcapable of recreating the cellular organization found in the outerneural retina and its interaction with the RPE. Here we describe anelectrospinning (ES) method used to form a fibrous scaffold directlyon living RPE cells, designed to direct PR outer segments toward anapical RPE membrane.Methods: RPE was isolated from adult mouse and human wereseeded onto glass coverslips coated with PDL and laminin andcultured to confluence. Polylactic Acid (PLA) was electrospundirectly onto samples and RPE cell survival analyzed. Scaffolds werethen seeded with a PR enriched retinal suspension and culturedtogether. Samples were fixed and stained for Bestrophin (RPE) andRecoverin (PR). Cell and scaffolds were imaged by confocalmicroscopy and environmental scanning electron microscopy(eSEM).Results: ES PLA fibers formed on living RPE were imaged by bothconfocal imaging and eSEM showed discrete fibers above livingcells. Following 5 days in culture, RPE were able to proliferate tonear confluence, but were still restricted to growth beneath the ESscaffold. PR seeded on these scaffolds were observed to form aseparate layer with cell bodies found above the fibers and processesextending along fibers towards the RPE layer mimicking the naturalorganization of the outer retina.Conclusions: By forming ES scaffolds directly above the RPE weare able to form a surface which restricts the growth of the RPE to amonolayer. This scaffold also provides a support for the seeding ofPR with pore sizes small enough to restrict the migration of both RPEand PR cell bodies but large enough to allow the extension of neuralprocesses and outer segments. Using this scaffold we have created amodel which recreates the 3D organization of the outer retina,allowing us to study the interaction of these two cells and therapiesfor the treatments of RD.Commercial Relationships: Karl E. Kador, None; Enrique Salero,None; Kristina R. Russano, None; Lung W. Lau, None; Jeffrey L.Goldberg, NoneSupport: RC1-EY020297; P30 EY014801Program Number: 1393 Poster Board Number: B0011Presentation Time: 8:30 AM - 10:15 AMRegenerative response of optic nerve axons while using aspecifically designed hydrogelArieh S. Solomon 1 , Anat Nitzan 1 , Moran Aviv 2 , Shmuel Einav 2 , EhudGazit 3 , Zvi Nevo 4 . 1 Eye Research Institute, Tel Aviv University,Ramat Gan, Israel; 2 Biomedical Engineering, Tel Aviv University,Tel Aviv, Israel; 3 Molecular Microbiology & Biotechnology, TelAviv University, Tel Aviv, Israel; 4 Human Molecular Genetics &Biochemistry, Tel Aviv University, Tel Aviv, Israel.Purpose: To provide a more permissive environment for axonalregeneration following injury to optic nerve.Regeneration of the CNSin mammals is very limited due to physical and chemical inhibitorsand barriers that react following optic nerve asault and the absence ofpositive cues that elicit and guide repair.Hydrogels are considered tobe good materials that support CNS repair due to high oxygen andnutrient permeabiltyand low interface tensions.Hyaluronic acid (HA)is a natural organic polymer used as a buliding block for hydrogelformation.It is used in medical surgical applications beeing similar toextracellular matrix (ECM) and beeing a biocompatible abiodegredable material.Peptide-based scaffolds represent anothervery import biocompatible material that can support cell growth.Methods: HA based composite containing self assembled,smallpeptides nano-tubes(FmocFF)was selected based on its netlike 3Dstructure.Cell culture analysis showed that the composite was nontoxixand allowed cell attachment.All animal study was carriedaccording to the approval of the University Commettee for AnimalResearch. The right eye of laboratory rat was cut within the meningesand sparing the vasculature.HA-FmocFF composite was implanted atthe lesion site.The cotrol group was implanted with 2% unmixedHA.Two months following the injury the optic ne were analysed byimmunohistochemical preparation. Longitudinal sections werelabeled with GAP43 antibody and neurotracer,cholera toxin subunitbeta, to determine axonal regeneration.Results: The HA-FcmocFF composite was found to be non-toxic andbiocompatible to the optic nerve, with no evidence ofdegeneration.Two months following injury, immunofluorescenceanalysis presented in most of the ONs a small number of axons thatwere found adjacent to the injury site.However, about a third of theONs treated with the HA-FmocFF composite showed a moresubstantial regeneration towards the optic chiasm.Conclusions: HA-FmocFF hydrogel appeared to contribute more toaxonal regeneration than hyaluronic acid alone.Partial regenerationcan be achieved with specifically designed hydrogels, which offer theregenerating axons a relatively free pathway and net-like 3d scaffoldto support and guide their growth beyond the lesion site. Further©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Nanotechnology and Regenerative Medicinestudy is needed, for longer periods of time of follow up, to elucidatethe stability of the regenerative process.Commercial Relationships: Arieh S. Solomon, None; Anat Nitzan,None; Moran Aviv, None; Shmuel Einav, None; Ehud Gazit,None; Zvi Nevo, NoneSupport: The Cuenca Institute for Research on AgingProgram Number: 1394 Poster Board Number: B0012Presentation Time: 8:30 AM - 10:15 AMBiocompatibility of helicoidal multi-lamellar features of RGDfunctionalizedsilk biomaterials for tissue engineering corneaLiqiang Wang, Huiling Guo, Ying Dong, Gaiping Du, Yifei Huang,Liang Jia. Ophthalmology, Chinese PLA General hospital, Beijing,China.Purpose: Corneal blindness accounts for nearly 10 million cases ofvision loss worldwide. Though corneal can be surgically removedand replaced with tissue from a deceased donor. Currently the onlyalternative to allograft tissue replacements is synthetickeratoprosthesis, however, these exhibits a relatively high hostrejection rate. Silk proteins represent a unique choice in biomaterialselection for tissue engineering and regenerative medicineapplications due to fibroin’s non-immunogenic response upon in vivoimplantation, controllable material degradation rates, tunable androbust mechanical properties. In this research, we implantedhelicoidal multi-lamellar features of RGD-functionalized silkbiomaterials in corneal stroma of rabbit, and evaluated thebiocompatibility of this new biomaterial.Methods: Implanted helicoidal multi-lamellar features of RGDfunctionalizedsilk biomaterials in corneal stroma of rabbit, setting upcontrol group at the same time. Observed the biomaterial under theslit lamp and Pentacam. Meanwhile, hematoxylin and eosin stain,immunofluorescence, scanning electron microscope were used to thecorneal at the 7d, 1m, 3m to evaluate the biocompatibility of this newbiomaterial.Results: The 2d, 4d, 7d, 30d, 90d rates of inflammatory reaction andnew vessels didn’t have obviously difference. The helicoidal multilamellarfeatures of RGD-functionalized silk biomaterials wereintegrity and transparent during the whole experiment. Thebiomaterials didn’t cause immunogenic response as well asdegradation in rabbit corneal stroma. The content of Collagen Iincreased over the 6 mouths, meanwhile the contects of Collagen IIIand fibronectin increased to a peak , then decreased gradully. Celladhesion, proliferation and morphology were detected at 3m byscanning electron microscope. The extracellular matrix depositedwell on the surface of the silk fibroin biomaterials, tightly adheredwith the biomaterial.Conclusions: Implanted helicoidal multi-lamellar features of RGDfunctionalizedsilk biomaterials in corneal stroma of rabbit, thebiomaterial had perfect biocompatibility with the corneal stroma. Thebiomaterial degradated slowly in the corneal tissue; cause lessinflammatory reaction or form of new vessels. This new biomaterialdidn’t cause immunological rejection as well.Commercial Relationships: Liqiang Wang, None; Huiling Guo,None; Ying Dong, None; Gaiping Du, None; Yifei Huang, None;Liang Jia, NoneSupport: NSFC funding 31271059Program Number: 1395 Poster Board Number: B0013Presentation Time: 8:30 AM - 10:15 AMA Novel Fish Scale Derived Scaffold for Ocular ReconstructionTine Possemiers 1, 2 , Nadia Zakaria 1, 2 , Veerle Van Gerwen 2 , Shih-Cheng Chen 3 , Horng J. Lai 3, 4 , Chien C. Lin 4 , Marie-José B.Tassignon 1, 2 . 1 Ophthalmology, Antwerp University Hospital,Edegem, Belgium; 2 University of Antwerp, Antwerp, Belgium;3 Research and Development, Aeon Astron Europe B.V., Leiden,Netherlands; 4 Research and Development, Body Organ BiomedicalCorp., Taipei, Taiwan.Purpose: The purpose of this study was to determine (1) thebiocompatibility of a decellularized fish scale derived scaffold withhuman corneal epithelial cells (CECs) in vitro, and (2) the optimalconditions for culturing primary CECs on the surface of the scaffold.Methods: First, Scanning Electron Microscopy (SEM) wasperformed to determine the ultra structure of the scaffold. Next,primary CECs were cultured on the scaffolds in CnT-20 medium.Cultures were evaluated by light microscopy andimmunofluorescence microscopy using antibodies against CK3/12,CK14, p63, FLT4 and ABCG2. To determine optimal cultureconditions, primary CECs were cultured on scaffolds using differentculture protocols: (A) Supplemental Hormonal Epithelial Medium(SHEM)+10% Foetal Bovine Serum (FBS) for 2 weeks, (B) CnT-20medium for 2 weeks, and (C) Combi-medium: CnT-20 for the firstweek and then switched to SHEM+10% FBS for the second week.Cultures were evaluated by phase contrast, SEM and TransmissionElectron Microscopy (TEM).Results: SEM revealed a patterned surface of the decellularizedscaffold (Fig 1). Phase contrast microscopy showed primary CECscultured on the scaffolds displayed a homogeneous epithelialmorphology (Fig 1). Immunofluorescence microscopy of culturedwhole mounts showed positive staining for CK3/12, CK14, p63,ABCG2 and FLT4. In the optimization experiments, TEM of primaryCECs cultured on scaffolds, following culture protocol A showed 3to 4 layers of cellular stratification with CECs in several stages ofdifferentiation. Cellular components and tight junctions were present.On SEM, the presence of numerous microvilli and cellular marginswere detected. Culture protocol B showed very few CECs adhering tothe scaffold, and a lack of desmosomes. Culture protocol C showedcellular stratification, clear desmosomes, but numerous largeintercellular spaces.Conclusions: We have been able to show that the decellularized fishscale derived scaffold is a biocompatible substrate with the potentialfor ophthalmic use as a bio-artificial cornea and that it is optimallycultured in SHEM+10%FBS for a period of 2 weeks for completecorneal epithelialization.A. SEM image of the scaffold. The characteristic patternedchannelling of the surface is observed. B. Phase contrast imageoverview of CEC growing within the patterned canals on the surfaceof the scaffold. ABCG2-FITC is shown in green and nuclei arestained with DAPI (blue).Commercial Relationships: Tine Possemiers, Aeon Astron EuropeB.V. (F); Nadia Zakaria, None; Veerle Van Gerwen, None; Shih-Cheng Chen, Aeon Astron Europe B.V. (E); Horng J. Lai, AeonAstron Europe BV (E); Chien C. Lin, None; Marie-José B.Tassignon, None©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Nanotechnology and Regenerative MedicineProgram Number: 1396 Poster Board Number: B0014Presentation Time: 8:30 AM - 10:15 AMAscl1 reprograms Müller glia into functional retinal neuronsJulia Pollak 1, 2 , Matthew Wilken 1, 3 , Yumi Ueki 1 , Kristin E. Cox 1 ,Russell J. Taylor 1 , Thomas A. Reh 1, 2 . 1 Department of BiologicalStructure, University of Washington, Seattle, WA; 2 Neurobiologyand Behavior Program, University of Washington, Seattle, WA;3 Molecular and Cellular Biology Program, University of Washington,Seattle, WA.Purpose: Teleost fish have a robust ability to regenerate retinalneurons from Müller glia cells. In contrast, mammalian Müller gliahave a limited regenerative response. Previous studies have shownthat the proneural transcription factor, Ascl1, is expressed in fishMüller glia early in the regeneration process; however, this factor isnot reactivated in mouse Müller glia after injury. Therefore, we testedwhether viral overexpression of Ascl1 could restore a neurogenicpotential to mouse Müller glia.Methods: Müller glia were grown as dissociated cultures or in retinalexplants and infected with lentiviruses encoding Ascl1 or GFP.Cultures and explants were examined for progenitor and retinalneuronal gene expression by RT-PCR, microarray analysis, andsingle cell immunohistochemistry. Functional analysis of cells wascarried out using ratiometric calcium imaging.Results: Müller glia expressing Ascl1 upregulated retinal progenitorspecificgenes, while downregulating glial genes by microarrayanalysis and RT-PCR. Müller glia-derived progenitors furtherdifferentiated into cells that exhibited neuronal morphologies andexpressed pan-neuronal and retinal subtype-specific neuronalmarkers, including Otx2, Islet1, Calretinin, and PKC. Müller glialderivedneurons responded to glutamate agonists with increases incalcium signaling and decreased responsiveness to purinergicagonists.Conclusions: These results indicate that a progenitor program can bereactivated in mammalian Müller glia through the re-expression ofthe proneural transcription factor Ascl1. These progenitorsdifferentiate into functional retinal neurons both in dissociatedcultures and in the intact retina, suggesting that Müller glia may be auseful source of endogenous replacement of mammalian retinalneurons in the future.Commercial Relationships: Julia Pollak, None; Matthew Wilken,None; Yumi Ueki, None; Kristin E. Cox, None; Russell J. Taylor,None; Thomas A. Reh, NoneSupport: NIH 1R01EY021482, NICHHD T32HD007183, NSFDGE-0718124, P30EY01730Program Number: 1397 Poster Board Number: B0015Presentation Time: 8:30 AM - 10:15 AMChitosan-Gelatin Biopolymers as Carrier Substrata for LimbalEpithelial Stem CellsTeresa Nieto-Miguel 1, 3 , Ana de la Mata 1, 3 , Marina López-Paniagua 1,3 , Sara Galindo 1, 3 , María R. Aguilar 2, 3 , Luis García-Fernández 2, 3 ,Blanca Vazquez 2, 3 , Julio San Román 2, 3 , Rosa M. Corrales 1, 3 ,Margarita Calonge 1, 3 . 1 Ocular Surface Group-IOBA, University ofValladolid, Valladolid, Spain; 2 Group of Biomaterials, Institute ofPolymer Science and Technology, CSIC, Madrid, Spain; 3 BiomedicalResearch Networking center in Bioengineering, Biomaterials andNanomedicine (CIBER-BBN), Valladolid and Madrid, Spain.Purpose: The aim of this work was to evaluate different semisyntheticbiopolymers based on chitosan (CH) and gelatin (G) aspotential in vitro carrier substrata for human cells derived from thelimbal niche, the so-called limbal epithelial stem cells (LESC).Methods: Semi-synthetic biopolymers were obtained using 85%deacetylated CH and different concentrations of pig skin G (0%,20%, 50% and 80%). Glutaraldehyde (0.50% wt) was added ascrosslinker. Human corneal epithelial cells (HCE) were cultured ontothe different membranes for several days. Cell adhesion, viability andproliferative capacity were analyzed using Calcein/EthDIII kit andAlamar blue test, respectively. LESC derived from human limbalexplants were expanded using a culture medium lacking componentsof animal origin. Tissue culture plastic was used as controlsubstratum. The expression of specific corneal epithelial cell markers(K3, K12, E-caherin, desmoplakin, and zonula occludens (ZO)-1) andLESC markers (K15 and ABCG2) was evaluated by real time RT-PCR and immunoflurescence microscopy.Results: None of the polymers tested were cytotoxic and HCE cellproliferation was higher when CH formulations were crosslinkedwith G. Expression levels of specific corneal epithelial markermRNAs KRT3, KRT12, E-caherin, desmoplakin, and ZO-1, as well asproteins K3 and K12 were better in HCE cells grown on CH-G 20:80membranes. Because of the sustained expression levels of thesemarkers by cells grown on that polymer, and because of the easyhandling of this substratum, CH-G 20:80 was chosen for thesubsequent expansion of human LESC. Cells derived from limbalexplants were successfully expanded on CH-G 20:80 membranes.The expression levels found for the LESC markers K15 and ABCG2in limbal cells expanded on CH-G 20:80 membranes were similar toor higher than those found in limbal cells grown onto the controlsubstratum.Conclusions: The successful growth of cells with a stem cell-likephenotype derived from limbal explants was achieved on a semisyntheticsubstratum in a medium free of non-human animalcomponents. CH-G 20:80 membranes were suitable for the expansionand maintenance of these LESC. These results strongly support theuse of polymers as alternative substrates for the transplantation ofcultivated limbal cells onto the corneal surface.Commercial Relationships: Teresa Nieto-Miguel, None; Ana de laMata, None; Marina López-Paniagua, None; Sara Galindo, None;María R. Aguilar, None; Luis García-Fernández, None; BlancaVazquez, None; Julio San Román, None; Rosa M. Corrales, None;Margarita Calonge, Allergan (C)Support: Instituto de Salud Carlos III: CIBER-BBN, Red TerCel,Centro en Red de Medicina Regenerativa y Terapia Celular deCastilla y León (Junta de Castilla y León).Program Number: 1398 Poster Board Number: B0016Presentation Time: 8:30 AM - 10:15 AMDifferent aligned multiwalled carbon nanotubes an their effectson cell viability and growth charactaristicsClaudia Etzkorn 1 , Sandra Johnen 1 , Frank Meissner 2 , Ingolf Endler 2 ,Peter Walter 1 . 1 Department of Ophthalmology, RWTH AachenUniversity, Aachen, Germany; 2 Frauenhofer Institute for CeramicTechnologies and Systems, Dresden, Germany.Purpose: Interfacing neuronal tissue with extracellularmicroelectrodes is a promising approach to regain functionality inpatients suffering from photoreceptor degeneration, e.g., retinitispigmentosa. To restore vision, prostheses that electrically stimulatesurviving retinal cells have already been developed. Optimization ofthe microelectrode properties related to charge transfer capacity andsignal to noise ratio can be achieved by nano modification, e.g.,coating with aligned multiwalled carbon nanotubes (MWCNTs).Aligned MWCNTs were synthesized with different catalyst particlesmixtures and investigated with regard to survival and proliferation ofdifferent cell types grown on these wafer pieces.Methods: Synthesis of aligned MWCNTs was carried out on 4-inchsilicon wafers by chemical vapour deposition. Length and©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Nanotechnology and Regenerative Medicinehomogeneity were improved with a SiO2 and an Al2O3 layer.Aligned MWCNT growth was achieved by iron particles (3 nm indiameter) or mixtures of iron-platinum and iron-titanium acting ascatalysts. Synthesis was carried out at atmospheric pressure usingArgon as carrier gas. Survival of L-929 cells and retinal precursor(R28) cells was estimated after direct contact with the wafer piecesusing fluorescein-diacetate/propidiumiodide life-dead assay. Cellgrowth was determined after indirect contact, which impliescultivation in medium pre-incubated with the respective wafer pieces,using a luminescent cell viability assay.Results: Both cell types, L-929 and R28, exhibited good proliferationand cluster formation properties on each tested MWCNT-coatedsilicon wafer, compared to non-coated pieces and a glass control. Cellviability ranged from 96.9% to 99.1%. However, cell survival onwafers generated with iron-platinum and iron-titanium catalystmixtures was better than on wafers generated with iron catalyst. Theindirect contact with pre-incubated medium had no significantinfluence on cell growth rates, measured in comparison with positivereference materials that show defined levels of toxicity.Conclusions: Different catalyst mixtures were tested to improve thesynthesis of aligned MWCNTs on silicon wafer pieces. Regardingcell viability, MWCNT-coating did not show any significant decreasein cell viability and is therefore a promising technology to improveinterfacing microelectrodes with retinal tissue.Commercial Relationships: Claudia Etzkorn, None; SandraJohnen, None; Frank Meissner, None; Ingolf Endler, None; PeterWalter, Novartis (R), Bayer (R), Second Sight (R), Bayer (F),Novartis (F)Program Number: 1399 Poster Board Number: B0017Presentation Time: 8:30 AM - 10:15 AMKeratin-chitosan membranes as scaffold for tissue engineering ofhuman corneaALVARO MEANA 1, 3 , Natalia Vázquez 1 , Manuel Chacón 1 , YolandaMenéndez-Menéndez 2 , Amaia Ferrero-Gutierrez 2 , Jesus Merayo-Lloves 1, 3 . 1 Superficie Ocular, Fundación de InvestigaciónOftalmológica, Oviedo, Spain; 2 Transplantes y Terapia Celular,Hospital Central de Asturias, Oviedo, Spain; 3 Universidad de Oviedo,Oviedo, Spain.Purpose: To study the attachment and growth of epithelial, stromaland endothelial cells of human cornea on keratin-chitosanmembranes. The end goal is to develop a bioengineered cornea basedon this material.Methods: 1) Cells. Eyes were attained from a local eye bank afterpenetrant-keratoplastic surgery, and cells were taken fromcorneoscleral-ring tissue. Epithelial, stromal and endothelial cellswere obtained from explants, 2-3 mm in diameter, of the limbal,stroma and descemet/endothelial regions. These cells were culturedin different media onto keratin-chitosan membranes, as well as on aplastic culture dish as control group. Cultures were examined byphase contrast microscopy.2) Membranes. Keratin-chitosan membranes were prepared aspreviously described in Tanabe et. al., 2002. Briefly, 7,15 mg/cm2 ofkeratin dialysate was diluted in three volumes of glacial acetic acid,which was then mixed with 10 p/p chitosan solution (2,5mg/ml in75% acetic acid solution) and 20 p/p glycerol. The solution was castinto a silicone mold and dried at 50 Celsius degrees for 36 hours.3) Histological methods. When cultured cells reached confluence,they were fixed with 4% paraformaldehyde, permeabilized withTriton X-100 and incubated at 4 Celsius degrees overnight withprimary antibodies (E-cadherin, Cytokeratin High Molecular Weight(CK), vimentin and N-cadherin). Immunolabeled cells werevisualized by indirect immunocytochemistry.Results: Epithelial, stromal and endothelial cells were able to attachand grow onto keratin-chitosan membranes. All the cells maintainedtheir morphology and cellular markers, both in the membrane and onthe culture plate.Epithelial cells exhibited a positive stain to CK and E-cadherin. Apositive vimentin stain was observed in all stromal cells, while inendothelial cells, a positive stain to vimentin and N-cadherin, butnegative to E-cadherin was found.Conclusions: Keratin-chitosan membranes have shown to be a goodscaffold for culturing epithelial, stromal and endothelial corneal cells;therefore, future applications of keratin-chitosan membranes may bedeveloped for reconstruction of the cornea.Commercial Relationships: ALVARO MEANA, None; NataliaVázquez, None; Manuel Chacón, None; Yolanda Menéndez-Menéndez, None; Amaia Ferrero-Gutierrez, None; Jesus Merayo-Lloves, Ferrara & Hijos SL (I)Program Number: 1400 Poster Board Number: B0018Presentation Time: 8:30 AM - 10:15 AMRestoration of light sensitivity to blind mice with red-shiftedchemical photoswitchesIvan Tochitsky 1 , Aleksandra Polosukhina 1 , Andrew Noblet 1 , DirkTrauner 2 , Richard H. Kramer 1 . 1 Molecular and Cell Biology,University of California, Berkeley, Berkeley, CA; 2 DepartmentChemie, Ludwig-Maximilians-Universität München, München,Germany.Purpose: Degenerative blinding diseases such as retinitis pigmentosaand age-related macular degeneration affect millions of patientsaround the world. These disorders cause the progressive loss of rodand cone photoreceptors in the retina, while sparing the remainingamacrine, bipolar and retinal ganglion cells. Visual function can berestored to a blind retina through gene delivery of light-sensitive ionchannels to the remaining retinal neurons. However, gene therapy canalso produce an unfavorable immune response or cause cancer. Itwould thus be highly desirable to develop more conventionalpharmaceutical means of restoring light sensitivity to a blind retina.Our goal is to develop and test such pharmacological therapies.Methods: We have created several small molecule chemical“photoswitches” that can be used to control the activity of neurons byreversibly blocking native ion channels in response to light. In orderto evaluate the ability of these photoswitches to restore lightsensitivity to blind mice, we have tested them in an rd1 mouse modelof retinitis pigmentosa. Our in vitro assays were carried out using amulti-electrode array (MEA). We also tested the restoration ofvisually guided behavior in vivo in several assays.Results: We have previously demonstrated that the photoswitchAAQ restores light responses to blind retinas in vitro and alsorestores the pupillary light reflex and light-aversive behaviors inblind mice.Now, we present the restoration of light sensitivity to blind mice invitro and in vivo with two improved photoswitch molecules, DENAQand BENAQ. Unlike AAQ, DENAQ and BENAQ do not require theuse of ultraviolet light and can be used to photosensitize the retina tovisible (blue-green) light. These red-shifted molecules photosensitizethe retinas of blind rd1 mice in vitro. The photoswitches persist up toseveral weeks in vivo and are well tolerated in the eye. DENAQ andBENAQ are selective for degenerated rather than healthy retinaltissue. Intravitreal injection of DENAQ also restores light sensitivityto rd1 mice in vivo in an exploratory locomotory behavioral assay.Conclusions: Red-shifted chemical photoswitches such as DENAQand BENAQ, and our pharmacological approach in general, holdgreat promise for restoring visual function in end-stage degenerativeblinding diseases.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Nanotechnology and Regenerative MedicineCommercial Relationships: Ivan Tochitsky, None; AleksandraPolosukhina, None; Andrew Noblet, None; Dirk Trauner, None;Richard H. Kramer, Photoswitch Biosciences (P)Support: NIH Grants EY018957, PN2 EY018241, EY003176;Beckman Foundation for Macular ResearchProgram Number: 1401 Poster Board Number: B0019Presentation Time: 8:30 AM - 10:15 AMIn Vivo Functional Comparison of Polarized and Non-polarizedhESC-RPE Cells Transplantation in RCS RatsLaura Liu 1, 2 , Biju B. Thomas 3 , Ramiro Ribeiro 3 , Alejandra Gonzalez-Calle 3 , Danhong Zhu 4, 3 , Padmaja B. Thomas 3 , Keng-Hung Lin 3 ,Yuntao Hu 3 , David R. Hinton 4, 3 , Mark S. Humayun 3 . 1 Cell andNeurobiology, University of Southern California, Los Angeles, CA;2 Ophthalmology, Chang Gung Memorial Hospitial, Taoyuan,Taiwan; 3 Ophthalmology, Doheny Eye Institute, Los Angeles, CA;4 Pathology, University of Southern California, Los Angeles, CA.Purpose: To evaluate the visual functional differences in dystrophicRoyal College of Surgeons (RCS) rats after transplantation of retinalpigment epithelial cells derived from H9 human embryonic stem cells(hESC-RPE) delivered as a polarized monolayer cell sheet on a thinparylene membrane or non- polarized cell suspension.Methods: For transplantation of polarized monolayer cell sheet,hESC-RPE cells were cultured for 3 weeks on parylene sheets; fortransplantation of cell suspension, hESC-RPE cells were cultured for3 weeks and digested into cell a suspension before implantation.Subretinal implantation of hESC-RPE /parylene (approximately 3000cells/implant, n=6) and subretinal injection of hESC-RPE cells (105 /2µl, n=6) was performed in 27 to 29 day old RCS rats. Six nonimplantedRCS rats served as controls. Prednisolone wasadministered through drinking water (0.002mg/liter) for the entireperiod of study. Post-surgical evaluation was performed usingspectral-domain optical coherence tomography (SD-OCT).Optokinetic head tracking (OHT) testing and luminance thresholdmeasurement from the superior colliculus (SC) were employed forvisual functional assessments at 2 months post-surgery.Results: Based on SD-OCT, preservation of the retinal layers in thetransplant area was observed in both implant groups. In hESC-RPE/parylene implanted animals, the head tracking duration washigher (4.06±0.76 sec/min) compared to the suspension injectiongroup (2.56±0.53 sec/min). SC recording demonstrated significantlylower (p=0.008) luminance threshold in the hESC-RPE/paryleneimplant group (-5.15±0.27 log cd/m 2 ) compared to the subretinalinjection group (-3.31±0.49 log cd/m 2 ). SC luminance threshold mapshowed good correlation with the location of the hESC-RPE/parylenein the retina.Conclusions: hESC-RPE when implanted as a monolayer or injectedas cell suspension demonstrated preservation of the retina in RCS ratsup to 2 months post-surgery. Better visual preservation was observedin the hESC-RPE/parylene implanted animals as evidenced by SCluminance threshold measurement and OHT testing. Our studysuggests that implantation of hESC-RPE/parylene may be a moredesirable therapeutic approach for disease conditions related to RPEdysfunction such as dry age-related macular degeneration.Commercial Relationships: Laura Liu, None; Biju B. Thomas,None; Ramiro Ribeiro, None; Alejandra Gonzalez-Calle, None;Danhong Zhu, None; Padmaja B. Thomas, None; Keng-HungLin, None; Yuntao Hu, None; David R. Hinton, RPT (I), RPT (P);Mark S. Humayun, Bausch & Lomb (F), Bausch & Lomb (C),Bausch & Lomb (P), Bausch & Lomb (R), Bausch & Lomb (S),Alcon (C), Alcon (R), Iridex (P), Iridex (R), Replenish (I), Replenish(C), Replenish (R), Replenish (S), Second Sight (F), Second Sight (I),Second Sight (C), Second Sight (P), Second Sight (R), Second Sight(S), Regenerative Patch Technologies (I), Regenerative PatchTechnologies (C)Support: California Institute for Regenerative Medicine (CIRM),NEI EY03040Program Number: 1402 Poster Board Number: B0020Presentation Time: 8:30 AM - 10:15 AMTissue Engineering of a Human Choroid Containing a VascularNetwork and MelanocytesStephanie Proulx 1, 2 , Marie Guimond 1 , Olivier Rochette-Drouin 1 ,Solange Landreville 1, 2 . 1 LOEX/CUO - Recherche, Centre derecherche du CHU, Quebec, QC, Canada; 2 Ophtalmologie, UniversitéLaval, Quebec, QC, Canada.Purpose: The goal of this study was to isolate three cell types fromthe choroid (fibroblasts, endothelial cells and melanocytes) in orderto reconstruct a choroid in vitro using the self-assembly approach oftissue engineering.Methods: Human choroids were incubated in dispase to removeretinal pigment epithelial cells, then in collagenase to dissociate thechoroidal cells. Vascular endothelial cells were isolated using CD31-coated magnetic beads, then cultured in EBM-2 growth medium. Theremaining cells were plated in growth media optimized for the cultureof melanocytes or fibroblasts. Culture purity was assessed usingimmunostainings (CD31, HMB45, vimentin, keratins 8/18). Toreconstruct the choroidal stroma, fibroblasts were cultured in thepresence of serum and ascorbic acid to promote extracellular matrix(ECM) assembly. Melanocytes and endothelial cells (primarycultures or GFP-HUVEC) were separately seeded on top of thestromal substitutes. Sheets were then stacked in order to sandwich theendothelial cells between the ECM sheets. Stromal substitutes wereanalysed by mass spectrometry and by immunostaining (collagens,proteoglycans). Development of vascular networks was observedthroughout time using the GFP-HUVEC cell line. Choroidalsubstitutes were further characterized by histology.Results: The technique used to isolate choroidal cells yielded purecultures of fibroblasts, melanocytes and vascular endothelial cells.The stromal substitutes engineered using the self-assembly approachwere composed of collagen (types I, VI, XII and XIV), proteoglycans(such as decorin, lumican) and other ECM proteins. Proteinexpression was confirmed using immunostaining. Endothelial cellsspontaneously assembled into tubular structures and vascularnetworks when cocultured within the fibroblast-containing ECMsheets. The seeded melanocytes adhered and survived on the stromalsubstitutes as confirmed by the presence of pigmented HMB45-positive cells with a dendritic morphology.Conclusions: This study shows that the self-assembly approach oftissue engineering can be used to reconstruct a choroid using nativecells. This model represents a unique tool to better understand thecrosstalk between the different choroidal cell types and cell-ECMinteractions.Commercial Relationships: Stephanie Proulx, None; MarieGuimond, None; Olivier Rochette-Drouin, None; SolangeLandreville, NoneSupport: Fondation HSS-HEJ, Centre de recherche du CHU, VisionResearch Network of the FRQSProgram Number: 1403 Poster Board Number: B0021Presentation Time: 8:30 AM - 10:15 AMA polymer-based interface restores light sensitivity in rat blindretinasMaurizio Mete 1 , Grazia Pertile 1 , Diego Ghezzi 2 , Maria RosaAntognazza 3 , Rita Maccarone 4 , Erica Lanzarini 3 , Nicola Martino 3 ,Silvia Bisti 4 , Guglielmo Lanzani 3 , Fabio Benfenati 2 . 1 Ophthalmology,©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Nanotechnology and Regenerative MedicineOspedale Sacro Cuore, Verona, Italy; 2 Department of Neuroscienceand Brain Technologies, Istituto Italiano di Tecnologia, Genova,Italy; 3 Center for Nanoscience and Technology, Istituto Italiano diTecnologia @POLIMI, Milano, Italy; 4 Department of BiomedicalTechnology, University of L'Aquila, L'Aquila, Italy.Purpose: Sight restoration is one of the new frontiers for prostheticdevices that enable the electrical stimulation of neurons. In particular,diseases that affect the retinal pigment epithelium and photoreceptorsbut preserve the inner retinal layers are preferential targets forimplantation of visual prostheses. We recently discovered thatprimary neurons can be successfully grown onto a transparentphotovoltaic organic polymer and electrically stimulated by light.This result encouraged us to test the efficacy of this method in retinasexplanted from albino rats with reproducibly induced photoreceptordegeneration due to light damage.Methods: We investigated the ability of the polymer layer to restorelight sensitivity in retinas explanted from albino rats with a lightinduceddegeneration of the photoreceptor layer. Acutely dissectedretinas were placed on the organic polymer in a sub-retinalconfiguration (i.e., external layers in contact with the polymer). Lightstimulation of the degenerate retina was observed by monitoringmulti-unit activity and field potentials with an extracellular electrodepositioned in the retinal ganglion cell layer.Results: Multi-unit activity recordings showed that a light stimulus16-fold lower than the safe limit for pulsed illumination elicitedintense spiking activity in degenerate retinas placed on polymercoatedsubstrates to levels indistinguishable from those recorded incontrol retinas. Moreover, to evaluate the efficiency of the interface,a dose-response analysis of spiking activity versus light intensitywere performed in degenerate retinas. Spiking activity was observedin degenerate retinas over the polymer with a response thresholdbelow 0.3 μW/mm2, a linear increase in a range corresponding todaylight irradiance, and a response saturation above 100 μW/mm2(considered the safe limit for chronic illumination). A 4-fold increasein the amplitude of the light response at saturation and a significantleft shift of the dose-response curves were obtained in retinas placedover the polymer-coated interface respect to degenerate retinas onglass substrates.Conclusions: Our finding indicate that the interface fully mimicksfunctional photoreceptors in activating the processing of the innerretina and is able to rescue normal light sensitivity. These resultsbroaden the possibility of developing a new generation of fullyorganic prosthetic devices for sub-retinal implants.Commercial Relationships: Maurizio Mete, None; Grazia Pertile,None; Diego Ghezzi, None; Maria Rosa Antognazza, None; RitaMaccarone, None; Erica Lanzarini, None; Nicola Martino, None;Silvia Bisti, None; Guglielmo Lanzani, None; Fabio Benfenati,NoneProgram Number: 1404 Poster Board Number: B0022Presentation Time: 8:30 AM - 10:15 AMReconstruction of the ocular surface with adipose-derived stemcells (ASCs)almudena del hierro 1 , Ana Boto 1 , Ignacio García Gomez 2 , MarianoGarcía-Arranz 3 , Alfredo Insausti García 1 , Félix Armada 1 .1 Oftalmología, Hospital La Paz, Madrid, Spain; 2 Medicine,University of Illinois, Chicago, IL; 3 Laboratorio de Terapia Celular,Hospital Universitario La Paz, Madrid, Spain.Purpose: To evaluate the ability and safety of the adipose-derivedstem cells (ASCs) as treatment to reconstruction of the cornealsurface of rabbits with experimentally induced limbal stem celldeficiency (LSCD).We compare two different procedures: perilimbarsubconjunctival injection versus application under an amnioticmembrane patch.Methods: Rabbit allogenic ASCs were isolated, cultured on plasticplates and identified by inmunostaining and flow cytometry. Limbalstem-cell deficiency (LSCD) was induced in 16 rabbits by alkaliburns and mechanical debridement. One week after the injury, eyeswere divided as follow: Group 1: 1A (n=8, left eyes) forsubconjuctival-limbal injection of 1.6 x 106 ASCs/400 μl distributedin four limbal quadrants and 1B (n=8, right eyes) for injection of 100μl of saline serum per quadrant; Group 2: 2A (n=8, Left eyes) forapplication of 1x106 ASCs under the stromal layer of amnioticmembrane and over the injured corneas and 2B (n=8, right eyes) fortransplantation of amniotic membrane. Each eye was examined undermicroscope every week before sacrifice. Rabbits were euthanizedafter 4 weeks and studied for inflammation (H&E staining), fibrosis(α-smooth muscle actin immunostaining), corneal inmunophenotype(Citokeratine 3) and inmunodetection of ASCs(GFP).Results: Incidence of perforations and calcifications was lower ineyes which had received ASCs by two procedures (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Nanotechnology and Regenerative Medicinemicroemulsions were chosen randomly and evaluated for oculartolerability. Twenty-three of the formulations showed no hyperemia,chemosis or discharge over the 5 days of QID dosing.Conclusions: Approximately 50% of the formulations were shown tobe well tolerated in this acute study. Current studies to quantify thecapability of these microemulsions to deliver various therapeuticmolecules to the posterior segment will yield valuable insighttowards the development of new topical formulations. Sinceinteractions are unique to the molecule and unpredictable, a largedata base is needed because the very presence of the drug candestabilize the emulsion. These results have formed the basis for ourformulation screening platform in which we intend to further expandour library to include more oil and surfactants.Commercial Relationships: Drew Wassel, Charlesson LLC (E),EyeCRO (E); Fadee Mondalek, Charlesson, LLC (E), EyeCRO (E);Alexander B. Quiambao, Charlesson, LLC (E), EyeCRO, LLC (E);Rafal Farjo, Charlesson LLC (E), EyeCRO LLC (E)Fig 1: W/S slopeProgram Number: 1406 Poster Board Number: B0024Presentation Time: 8:30 AM - 10:15 AMEarly data on the effect of continuous positive airway pressure(CPAP) on intraocular pressure (IOP) changes measured by acontact lens sensor (CLS) in patients with obstructive sleep apneasyndrome (OSAS) with and without primary open-angleglaucoma (POAG)Jessica V. Jasien 1 , Rene Goedkoop 2 , Cinthi Pillai 1 , Sonja Simon-Zoula 2 , Robert Ritch 1, 3 . 1 Einhorn Clinical Research Center, NewYork Eye and Ear Infirmary, New York, NY; 2 Sensimed AG,Lausanne, Switzerland; 3 New York Medical College, Valhalla, NY.Purpose: To investigate the effect of CPAP on IOP fluctuationsassociated with changes from wake to sleep (W/S) and sleep to wake(S/W) in patients both with and without POAG suffering from OSAS,measured by continuous recording using a CLS.Methods: In this single-center, prospective, exploratory, open-labelstudy, POAG and non-POAG patients with moderate to severe OSASunderwent continuous 24-hour IOP recording using Triggerfish®(Sensimed AG, Lausanne, Switzerland) in 2 sessions, 7 days apart, inthe same eye. W/S and S/W slopes for CLS were computed by fittinglinear regression to measurements from 1 hour before transition to 1hour after. Slopes reported are changes in millivolts/hour. Effect sizeswere computed by Cohen’s d.Results: We analyzed 8 OSAS patients, 4 with and 4 without POAG(mean age 64.3±7.8 years, 75% men). With CPAP, W/S slope forboth groups was similar (44.4±34.8 and 42.2±40.7, respectively).Without CPAP, the W/S slope increased to 52.3±26.4 for POAGeyes, and decreased to 35.8±43.0 for non-POAG eyes (Fig 1). Thusthe effect size of the difference between the groups when not usingCPAP was 0.46 and only 0.06 when using CPAP, W/S slopebecoming more moderate with CPAP in POAG and the reverse innon-POAG. Similarly, the S/W slope was more moderate in POAGwith CPAP than without (-15.5±10.4 vs -24.4±29.1); in non-POAGthe difference was once again reversed (with CPAP -36.1±21.1 andwithout -9.1±40.9 (Fig 2). The effect size of the difference betweenthe groups without CPAP was 0.43 and with CPAP was 1.24. Allslopes were in the expected direction (positive for W/S , indicating anincrease in CLS IOP fluctuations; and visa versa for S/W).Conclusions: POAG patients with CPAP had a slower increase ofIOP going from W/S and a slower decrease going from S/W thanwithout CPAP. A larger trial is warranted to substantiate thebeneficial effects of CPAP in POAG patients with OSAS.Fig 2: S/W slope. For a positive slope, higher (more positive) is moreextreme and for a negative slope, lower (more negative) is moreextreme; i.e. regardless of sign, larger absolute numbers representsteeper slopes than smaller absolute numbers.Commercial Relationships: Jessica V. Jasien, None; ReneGoedkoop, Sensimed AG (E); Cinthi Pillai, None; Sonja Simon-Zoula, Sensimed AG (E); Robert Ritch, NoneClinical Trial: NCT01560975Program Number: 1407 Poster Board Number: B0025Presentation Time: 8:30 AM - 10:15 AMOptimized cell-handling of human embryonic stem cells in thedifferentiation of photoreceptor precursor cellsChristopher R. Laver, Anat Yanai, Aaron Joe, Ishaq Ahmedviringipurampeer, Xia Wang, Andrew Metcalfe, Cheryl Y. Gregory-Evans, Kevin Gregory-Evans. Ophthalmology & Visual Sciences,University of British Columbia, Vancouver, BC, Canada.Purpose: A pressing need in regenerative medicine focused ontreating common blinding diseases such as age-related maculardegeneration and retinitis pigmentosa is the efficient production oflarge numbers of human retinal precursor cells. We proposed tooptimize retinal differentiation protocols for human embryonic stemcells (hESCs) by improving cell handling.Methods: To improve efficiency we firstly focused on the productionof just one retinal precursor cell type (photoreceptor precursor cells,PPCs) rather than the production of a range of retinal cells.Combining information from a number of previous studies, inparticular the use of feeder-free culture media and taurine plus triiodothyroninesupplements, we then assessed the values of usingsize-controlled embryoid bodies (EBs) and negative cell selection (toremove embryonic antigen-4 positive hESCs).Results: Using size controlled 1000-cell EBs, significantimprovements were made over previous studies in that 77% CRX+ve©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Nanotechnology and Regenerative MedicinePPCs (validated via FACS) could be produced in just 17 days. Thiscould be increased to 93% PPCs through the added step of negativecell selection. The PPC-phenotype of these cells was furthersupported by their expression of BLIMP1.Conclusions: We have developed a rapid, efficient method forproducing large numbers of PPCs for future preclinical studies incommon blinding conditions.Commercial Relationships: Christopher R. Laver, None; AnatYanai, None; Aaron Joe, None; Ishaq Ahmed viringipurampeer,None; Xia Wang, None; Andrew Metcalfe, None; Cheryl Y.Gregory-Evans, None; Kevin Gregory-Evans, NoneProgram Number: 1408 Poster Board Number: B0026Presentation Time: 8:30 AM - 10:15 AMMesenchymal stem cell based ex vivo gene therapy providesenhanced neuroprotection in X-linked retinoschisis mouse modelAMA E. Bashar, Andrew Metcalfe, Ishaq Ahmed viringipurampeer,Cheryl Y. Gregory-Evans, Orson L. Moritz, Kevin Gregory-Evans.Ophthalmology & Visual Sciences, University of British Columbia,Vancouver, BC, Canada.Purpose: X-linked retinoschisis (XLRS) is a juvenile-onset maculardegeneration caused by haplo-insufficiency of a cell adhesion proteinretinoschisin (RS1), secreted by photoreceptors and bipolar cells.RS1 mutations can lead to secretion of non-functional proteins, thus,as loss-of-function mutations are more amenable to gene therapy,XLRS is an excellent candidate for ex vivo gene therapy. Here, wereport the structural and functional benefits observed afterintravitreous delivery of MSCs genetically modified to secreteretinoschisin in the retina of our XLRS mouse model.Methods: MSCs were harvested from syngeneic RS1KO mouseadipose tissue and transfected via electroporation with a plasmidcontaining human RS1 cDNA under a CMV promoter. Transfectedcells were selected using geneticin for two weeks and then assayedby ELISA for retinoschisin synthesis. 200,000 genetically modifiedMSCs, secreting 8 ng/million cells/day, were injected into thevitreous cavity of the RS1KO mice at the age of post natal day 21.The control groups received either unmodified MSCs or shaminjection. Electroretinogram (ERG), optokinetic tracking response(OKT) and histology data were recorded for all cohorts at 2, 4 and 8weeks post injection time points.Results: Enhanced neuroprotection and inner nuclear layer tissueintegrity were observed in retinas which received modified MSCsinjection compare to the sham injection at all three time points.Lower a/b wave ratio in dark-adapted maximal ERG response andincreased spatial frequency thresholds in OKT highlightedimprovement in preserving vision in this group compared to others.These functional benefits were correlated with increased structuralpreservation of the inner nuclear layer.Conclusions: Intravitreal delivery of genetically modified MSCssecreting retinoschisin showed promising results in our XLRS mousemodel. Our previously reported magnetic targeting of systemicallydelivered cells would reduce the collateral damage caused by theintraocular injection of these cells, making this ex vivo approachsafer, less-invasive, and repeatable; future investigation will focus onthis delivery approach.Commercial Relationships: AMA E. Bashar, None; AndrewMetcalfe, None; Ishaq Ahmed viringipurampeer, None; Cheryl Y.Gregory-Evans, None; Orson L. Moritz, None; Kevin Gregory-Evans, NoneSupport: CIHRHistological correlation of superior collicus response to lightpreserved by transplantation of hESC derived RPE monolayer inRCS ratsBiju B. Thomas 1 , Padmaja B. Thomas 1 , Laura Liu 2, 3 , Yuntao Hu 1 ,Danhong Zhu 1, 4 , Dennis O. Clegg 5 , David R. Hinton 1, 4 , Mark S.Humayun 1 . 1 Ophthalmology, Doheny Eye Institute, Los Angeles,CA; 2 Cell and Neurobiology, University of Southern California, LosAngeles, CA; 3 Ophthalmology, Chang Gung Memorial Hospital,Taoyuan, Taiwan; 4 Pathology, University of Southern California, LosAngeles, CA; 5 Center for Stem Cell Biology and Engineering,University of California-Santa Barbara, Santa Barbara, CA.Purpose: Histological evaluation of the retina of Royal College ofSurgeon (RCS) rats that received subretinal implantation of humanembryonic stem cell (hESC) derived retinal pigment epithelial (RPE)cells cultured on the biocompatible polymer parylene as a monolayerand its correlation with visual functional preservation in the superiorcolliculus (SC).Methods: Polarized hESC-RPE cells (H9) were cultured for 4 weekson parylene sheets. Rectangular pieces (0.4mm x 0.9mm) of parylenewere seeded with RPE cells, which formed a confluent monolayer.These grafts were implanted into the subretinal space of postnatal (P)28 RCS rats (n=16). All animals were maintained underimmunosuppression for the entire period of study. Histologicalevaluation was performed based on immunostaining. In selectedanimals, electrophysiological mapping of the SC using various lightintensity stimulus was performed at 2 months post-implantation.Results: At P90, immunostaining demonstrated robust photoreceptor(rod and or cone) survival in an area overlying the hESC-RPEimplant. In non-treated dystrophic RCS eyes, photoreceptor survivalat this age was negligible. Long-term (6 months post-implantation)evaluation confirmed the preservation of rod and cone photoreceptorsin the implant area. In all hESC-RPE implanted animals, at thresholdstimulus level, responses in the SC could be recorded only from anarea corresponding to the implant location in the eye.Immunostaining revealed better rod preservation (4.25±0.59 layers)among rats that responded to low level light stimulation (-5.4 logcd/m 2 ). Rats that responded only at higher light stimulation (>-4.4 logcd/m 2 ) showed less rod survival (2.25±1.1 layers) and majority ofthese rods were inferior in appearance. No such differences betweentwo luminance measurement groups were observed in the degree ofcone survival.Conclusions: hESC-RPE cultured on parylene substrates implantedinto the RCS retina is capable of preserving rod and conephotoreceptors up to P180. Histological correlation of SC luminancemeasurement suggests preservation of functional rod and conephotoreceptors. The results support the use of such implants fortherapeutic approaches aimed at slowing the progression of outerretinal dystrophies such as age-related macular degeneration (AMD).Commercial Relationships: Biju B. Thomas, None; Padmaja B.Thomas, None; Laura Liu, None; Yuntao Hu, None; DanhongZhu, None; Dennis O. Clegg, Regenerative Patch Technologies (I);David R. Hinton, RPT (I), RPT (P); Mark S. Humayun, Bausch &Lomb (F), Bausch & Lomb (C), Bausch & Lomb (P), Bausch &Lomb (R), Bausch & Lomb (S), Alcon (C), Alcon (R), Iridex (P),Iridex (R), Replenish (I), Replenish (C), Replenish (R), Replenish(S), Second Sight (F), Second Sight (I), Second Sight (C), SecondSight (P), Second Sight (R), Second Sight (S), Regenerative PatchTechnologies (I), Regenerative Patch Technologies (C)Support: California Institute for Regenerative Medicine (CIRM),NEI EY03040Program Number: 1409 Poster Board Number: B0027Presentation Time: 8:30 AM - 10:15 AMProgram Number: 1410 Poster Board Number: B0028Presentation Time: 8:30 AM - 10:15 AM©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Nanotechnology and Regenerative MedicineGeneration of engineered human corneal lenticule withepithelium for selective allograftMAN-IL HUH 1, 2 , Eun-Jin Yeo 1 , Jun Hun Lee 1 , Hong-Kyun Kim 1, 2 .1 Ophthalmology, Kyungpook national university school of medicine,Daegu, Republic of Korea; 2 Joint Institute for RegenerativeMedicine, Kyungpook National University Hospital, Daegu,Republic of Korea.Purpose: The aim of this study was to evaluate utility value ofhuman corneal lenticule produced after refractive surgery withRelEx TM (Carl Zeiss). We investigated whether the corneal lenticuleis suitable for a scaffold which reconstituting cornea includingepithelium. In this study, four decellularization protocols wereevaluated to determine suitability as the scaffold, and we testeddecellularized lenticules to be useful for multilayered epitheliumsheet for selective allograft.Methods: To apply corneal lenticule to selective allograft, we madecorneal lenticule decellularized by 0.25% trypsin-EDTA, 0.1 % SDS,or 0.1 % Triton X-100 in hypotonic Tris buffer (10mM, pH 7.6). Andthen remained nucleic acid and cell morphology were visualized byDAPI and vimentin staining. Furthermore, the lenticules werecryosectioned with 10 mm thickness, and images were captured by afluorescence microscope. To test attachment of epithelial cells on thelenticule, the lenticules were coated with various extracellularmatrices (ECM) composing basement membrane, such as type Icollagen, type IV collagen, or laminin. We used CnT-20 media forconfluent monolayer culture of epithelial cells on lenticules, andCnT-30 media containing hormonal additives were used for makingmultilayered epithelium. The epithelial sheets were analyzed byhistological methods.Results: Histological analysis of the decellularized corneal lenticulesshowed that trypsin-EDTA treated group is completely acellular andhas not detected structure protein such as vimentin differently withother groups. Monolayer culture of epithelial cells showed thatconfluent culture and epithelial marker expressions are achieved byusing CnT-20 media. Results of immunohistochemistry showed thatmultilayered epithelium on the lenticule is composed of cellsexpressing marker such as CK15, and CK3/12 and that is similar withresult on amniotic membrane and fibrin gel.Conclusions: The lenticule from human corneal stroma wassuccessfully decellularized by 0.25% trypsin-EDTA in hypotonic Trisbuffer. And generation of multilayered epithelial sheet using thelenticule was successfully achieved by using CnT-20 and CnT30containing hormonal additives. Collectively, data show thatdecellularized lenticule has potential for use in selective allograft tochange epithelium and can be employed as a scaffold.Commercial Relationships: MAN-IL HUH, None; Eun-Jin Yeo,None; Jun Hun Lee, None; Hong-Kyun Kim, NoneSupport: KRF 2012R1A1A1010163249 Nanotechnology and Regenerative MedicineMonday, May 06, 2013 11:00 AM-12:45 PMTCC 305 Paper SessionProgram #/Board # Range: 1761-1767Organizing Section: Nanotechnology and Regenerative MedicineProgram Number: 1761Presentation Time: 11:00 AM - 11:15 AMPotentiation of bipolar cell GABA A receptors by a photoisomerizablecompoundLan Yue 1, 2 , Michal Pawlowski 3 , Karol S. Bruzik 3 , David R.Pepperberg 1, 2 . 1 Ophthalmology and Visual Sciences, University ofIllinois at Chicago, Chicago, IL; 2 Bioengineering, University ofIllinois at Chicago, Chicago, IL; 3 Medicinal Chemistry andPharmacognosy, University of Illinois at Chicago, Chicago, IL.Purpose: Photochemical switches that regulate the activity ofpostsynaptic membrane receptors of inner retinal neurons represent apossible approach to vision restoration in photoreceptor degenerativediseases such as age-related macular degeneration. MPC088, apropofol analog that incorporates a photo-isomerizable azobenzenegroup, exhibits isomer-specific and thus photo-controllablepotentiation of GABA A receptors of retinal ganglion cells (ref. 1). Inthe present study we asked whether GABA A receptors of retinalbipolar cells (RBCs) exhibit controllability by MPC088.Methods: Single, isolated RBCs were prepared from retinas of 6-16week-old Sprague-Dawley rats (1,2) and identified by cellmorphology. Membrane currents were recorded by whole-cellvoltage clamp (3). Trans-MPC088, the isomer with pronouncedactivity at ganglion cells (1), was converted to the cis-isomer byexposure to UV light (wavelengths near 365 nm) (1). Ringer solutioncontaining GABA and trans- or cis-MPC088 was presented to theRBC by gravity-driven superfusion. Test solutions also containedTPMPA to inhibit RBC GABA A -ρ (GABA C ) receptors.Results: Trans-MPC088 exhibited robust concentration-dependentpotentiation of RBC GABA A receptors. With co-applied 100 μMTPMPA, 5 μM trans-MPC088 produced an ~1.7-fold enhancementof the GABA A component of the 10 μM GABA response (n=3). Thepotentiation factor increased to ~2.4-fold with 30 μM trans-MPC088(n=9), and the potentiated GABA response was nearly eliminated bypicrotoxin (100 μM), a GABA A channel blocker (~95% inhibition,n=3). UV-illuminated (thus, cis-dominant) 30 μM MPC088 produceda potentiation (~1.3-fold, n=5) significantly lower (p=0.0003) thanthat determined with the trans-isomer.Conclusions: MPC088 exhibits isomer-dependent potentiation ofRBC GABA A receptors, offering the possibility of photo-control at apost-photoreceptor stage presynaptic to ganglion cells (4). The resultsencourage development of MPC088-like structures that possessoptimized wavelength sensitivity and relaxation kinetics, and that, viaan incorporated affinity reagent, can target GABA A receptors onspecific retinal cell types. (1) Yue et al. (2012) Nature Comm. 3:1095(doi: 10.1038.ncomms2094). (2) Ramsey et al. (2007) Exper. EyeRes. 85:413-422. (3) Yue et al. (2011) Invest. Ophthalmol. Vis. Sci.52:2497-2509. (4) Lagali et al. (2008) Nature Neurosci. 11:667-675.Commercial Relationships: Lan Yue, University of Illinois atChicago (P); Michal Pawlowski, University of Illinois at Chicago(P); Karol S. Bruzik, University of Illinois at Chicago (P); David R.Pepperberg, University of Illinois at Chicago (P)Support: NIH grants EY016094 and EY001792; Daniel F. and AdaL. Rice Foundation (Skokie, IL); Beckman Initiative for MacularResearch (Los Angeles, CA); American Health AssistanceFoundation (Clarksburg, MD); Research to Prevent Blindness (NewYork, NY); and Award UL1RR029879 from the University of Illinoisat Chicago Center for Clinical and Translational Science (CCTS).Program Number: 1762Presentation Time: 11:15 AM - 11:30 AMInsulin-like Growth Factor Binding Protein-like 1 (IGFBPL1) asa Potent Regulator of Optic Nerve RegenerationChenying Guo 1 , Kin-Sang Cho 1 , Kissaou T. Tchedre 1 , Jie Ma 1 ,Christian Antolik 1 , Dong F. Chen 1, 2 . 1 Ophthalmology, HarvardMedical School, Schepens Eye Research Institute, Boston, MA; 2 VABoston Healthcare System, Boston, MA.Purpose: We have recently reported the identification of the insulinlikegrowth factor binding protein like protein 1 (IGFBPL1) whichfunctions through mediating the bioactivity of IGF-1 to promote thegrowth of retinal ganglion cell (RGC) axons. To further elucidate the©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Nanotechnology and Regenerative Medicinephysiological function and signaling events induced by IGFBPL1, weexamined igfbpl1 knockout (ibl-/-) mice.Methods: To evaluate the functional significance of IGFBPL1 invivo, we generated ibl-/- mice and quantified the numbers of RGCsand their axons in wild-type (WT) and ibl-/- mice. Taking advantageof our ability to purify RGCs, we also assessed the survival and axongrowth potential of RGCs of WT and ibl-/- mice in culture. Theeffect of IGFBPL1 in optic nerve regeneration in the adult wasinvestigated further by injecting IGFBPL1 intravitreally into adultmice undergone optic nerve crush injury. Regeneration of the opticnerve was assessed quantitatively by counting RGC axons that wereanterogradely labeled with cholera toxin B subunit (CTB). Thesignaling events underlying IGFBPL1 activity were investigatedusing RGCs cultures isolated from postnatal day 10 (P10) retinas.Changes of intracellular Ca2+ concentrations and activations of IGF-1 mediated signals, including CREB, Akt, and mTOR wereexamined, using Ca2+ imaging and Western blot analysis.Results: ibl-/- mice displayed over 50% reduction in axon numbersin the optic nerves and diminished axonal outgrowth in culturedRGCs purified from the ibl-/- mice, as compared to WT littermatecontrols. The number of RGCs in ibl-/- mice, however, was onlyslightly reduced as compared to WT mice. Intravitreal injection ofIGFBPL1, in contrast, significantly enhanced optic nerveregeneration post optic nerve injury. Furthermore, we observed thatIGFBPL1 promoted calcium signaling and the activation of mTOR inRGCs to promote axonal growth.Conclusions: IGFBPL1 is a potent regulator of optic nerve growthand regeneration, which functions through IGF-I signaling topromote mTOR phosphorylation and intracellular Ca2+ signaling, thetwo key downstream effectors that control axonal growth. Theseresults provide novel insights into the molecular events underlyingthe control of RGC axonal growth and suggest potential newtherapeutic strategies for optic nerve protection and regeneration orrepair.Commercial Relationships: Chenying Guo, Schepens EyeResearch Institute (P); Kin-Sang Cho, None; Kissaou T. Tchedre,Menicon, Co. Ltd (E); Jie Ma, None; Christian Antolik, None;Dong F. Chen, GlaxoSmithKline (F), Patent/Schepens Eye ResearchInstitute (P)Support: Molecular Bases of Eye Disease, NEI EY017641, NationalInstitute of Drug Abuse DA024803, the Department of VeteransAffairs 1I01RX000110, the Department of Defense W23RYX-9104-N603, W81XWH-09-2-0091, W81XWH-11-1-0477groups and subretinally implanted with gelatin films without the cells(Group A, n=9) or gelatin films with confluent hESC-RPE cells(Group B, n=9). One week, one month and three months postimplantationOCT scanning and histological examinations wereperformed.Results: The gelatin without the cells did not degrade in subretinalspace up to 3 months after implantation (Fig.1). Degradation of thegelatin with hESC-RPE cells was observed at 3 months afterimplantation (Fig.2). However, the hESC-RPE cells maintainedmonolayer RPE morphology and were RPE65 positive.Conclusions: Degradation of subretinally implanted gelatin scaffoldsis cell-dependent and may be related to the release of proteolyticenzymes or other factors by hESC-RPE cells.Fig.1 Gelatin film (without cells) did not degrade after subretinalimplantation 3 months.Program Number: 1763Presentation Time: 11:30 AM - 11:45 AMSubretinal Implantation of Gelatin Films with Stem CellsDerived RPE in RatsYuntao Hu 1, 3 , Kenrick Kuwahara 1 , Padmaja B. Thomas 1 , BrunoDiniz 1, 4 , Ramiro Ribeiro 1, 5 , Ashish K. Ahuja 1 , Sherry T. Hikita 2 ,Lincoln V. Johnson 2 , Biju B. Thomas 1 , Mark S. Humayun 1 . 1 DohenyEye Institute, Keck School of Medicine of the University of SouthernCalifornia, Los Angeles, CA; 2 Center for Stem Cell Biology andEngineering, University of California-Santa Barbara, Santa Barbara,CA; 3 Ophthalmolgy, Peking University Third Hospital, Beijing,China; 4 Retina Sector, Universidade Federal de Sao Paulo, Sao Paulo,Belize; 5 Hospital Evangelico de Curitiba, FEMPAR, Curitiba, Belize.Purpose: Human embryonic stem cell-derived retinal pigmentepithelial (hESC-RPE) cells were cultured on gelatin films andimplanted into the subetinal space in rats in order to monitor postimplantationcell fate and gelatin degradation rate.Methods: Monolayer cultures of hESC-RPE cells were cultured on27 μm thick gelatin films. RCS rats were randomly divided into twoFig.2 The hESC-RPE cells maintained monolayer on gelatin filmafter subretinal implantation 3 months. Gelatin degraded partly nearto the hESC-RPE cells.Commercial Relationships: Yuntao Hu, None; KenrickKuwahara, None; Padmaja B. Thomas, None; Bruno Diniz, None;Ramiro Ribeiro, None; Ashish K. Ahuja, None; Sherry T. Hikita,None; Lincoln V. Johnson, None; Biju B. Thomas, None; Mark S.Humayun, Bausch & Lomb (F), Bausch & Lomb (C), Bausch &Lomb (P), Bausch & Lomb (R), Bausch & Lomb (S), Alcon (C),Alcon (R), Iridex (P), Iridex (R), Replenish (I), Replenish (C),Replenish (R), Replenish (S), Second Sight (F), Second Sight (I),Second Sight (C), Second Sight (P), Second Sight (R), Second Sight(S), Regenerative Patch Technologies (I), Regenerative PatchTechnologies (C)Support: This research was supported by CIRM DRI-01444,Research to Prevent Blindness, and NEI grant EY03040.Program Number: 1764Presentation Time: 11:45 AM - 12:00 PM©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Nanotechnology and Regenerative MedicineRPE for photoreceptor regeneration in mouse eyeShu-Zhen Wang, Run-Tao Yan. Ophthalmology, Univ of Alabama atBirmingham, Birmingham, AL.Purpose: Current research on producing mammalian photoreceptors,as regenerative medicine in general, focuses on ES cells and iPScells. This project studies a different approach - tapping the naturallyoccurring wound healing mechanism of RPE for photoreceptorregeneration. Previous experiments found extra photoreceptor cells intransgenic mice generated with a DNA cassette expressing ngn1 fromRPE promoter PVMD2. This study aims to determine RPE as asource of the extra photoreceptor cells.Methods: Transgenic mice were generated with a DNA construct, inwhich RPE bestrophin-1 promoter (PVMD2) would drive theexpression of ngn1, a regulatory gene with pro-photoreceptoractivity. Eyes were analyzed for the presence of cells in “RPE-tophotoreceptor”transition with histology and immunohistology.Results: Cells in “RPE-to-photoreceptor” transition were detected intransgenic eyes. Some darkly pigmented cells, as well as discretecells in the RPE, were recoverin+. Morphologically, these recoverin+cells were more similar to RPE cells than to photoreceptor cells. Inaddition to their pigment granules, which otherwise are typicallypresent in RPE cells, these recoverin+ cells displayed a nucleus thatappeared (a) round (as in RPE cells) vs. elongated (as inphotoreceptors), (b) large vs. compact, and (c) contained severaldiscrete, brightly fluorescent spots of DAPI staining. Notably, in a 9-month-old PVMD2-ngn1 animal, anti-recoverin immunoreactivitywas detected in essentially all cells within a particular region of theRPE. Nearby, a layer, reminiscent of an emerging extra photoreceptorlayer, was present in the subretinal space. Cells in this layer showedpigment granules, as seen in RPE cells. Yet, they were recoverin+and displayed an elongated cell body. At the tip of the emerginglayer, a well defined extra photoreceptor layer was apparent.Immunostaining showed that the RPE in the region became negativefor Otx2, a homeodomain factor involved in maintaining RPE vs.retina identity and no longer expressed in RPE-to-retinatransdifferentiation in newt.Conclusions: These results suggest that RPE-to-photoreceptorreprogramming was occurring in transgenic mouse and, thus, RPEwas a source of the extra photoreceptor cells present in the transgenicmouse. The presence of cells in “RPE-to-photoreceptor” transition ina 9-month-old transgenic animal suggests that the reprogrammingcould occur in aged eyes.Commercial Relationships: Shu-Zhen Wang, None; Run-TaoYan, NoneSupport: NIH/NEI grant EY011640, EyeSight Foundation, andResearch to Prevent Blindness.Program Number: 1765Presentation Time: 12:00 PM - 12:15 PMConjugation of antibody on biocompatible nanoparticles fortargeted drug deliveryJin Zhang 1, 2 , Wai Hei Tse 1 , William G. Hodge 2 . 1 Chemical andBiochemical Engineering, University of Western Ontario, London,ON, Canada; 2 Ivey Eye Institute,, London, ON, Canada.Purpose: This project aims at the development a targeted drugdelivery system by usinghydrogel nanospheres for reducing the frequency of injection andfollow-up diagnosis for age-related macular degeneration (AMD).Methods: Bio-compatible luminescent nanoparticles (NPs) act asdrug carriers for the targeted delivery to inhibit the growth ofabnormal blood vessel in the area of macula. The nanoparticles (NPs)as drug carriers will maintain the bioactivity of the conjugated antihumanIgG (Fab specific) antibody, and keep the local concentrationof the anti-body at a desire level, which may result in the efficienttreatment for AMD in a sustainable manner. Meanwhile, the newluminescent NPs as drug carriers can be identified easily by applyingoptical coherence tomography (OCT), a non-invasive diagnostic tool.Results: In this study, CdSe Qquantum dots (QDs) were firstmodified with a zinc sulfide shell to solubilize the QD in water. TheQDs-loaded gelatin-basedr nanoparticles (QDs-GNPs) are furtherdeveloped to be conjugated with anti-human IgG antibody which isable to drive the nano-carriers to the cell with high expression ofVEGF, and block VEGF at its original site in the extracellular space.Gelatin is a colorless and translucent natural polymer derived fromcollagen of animals’ bones and skin. It has been used in food andpharmaceutical industry because of its multi-functional groups (-COO, -NH2), biodegradability, and unique gel-forming ability. Ourrecent results demonstrate that gelatin NPs can keep the loadedprotein drug bioactivity in vitro during the long releasing period (t =>20 days). In addition, we show the multifunctional modification onthe surface of cationic nanoparticles, e.g. gelatin NPs, chitosan NPs,and silica NPs to recognize certain protein on the surface of cells.Our results indicate that surface modified QD-GNPs do not imposeany toxic effect on the cell lines.Conclusions: This system will allow us to evaluate the nanosystemfor imaging and treatment of AMD in vitro.Commercial Relationships: Jin Zhang, None; Wai Hei Tse, None;William G. Hodge, NoneSupport: NSERCProgram Number: 1766Presentation Time: 12:15 PM - 12:30 PMFace Detection using the Argus® II Retinal Prosthesis SystemPaulo E. Stanga 1, 2 , Jose A. Sahel 5, 4 , Saddek Mohand-Said 4, 5 , LyndondaCruz 6 , Avi Caspi 3 , Francesco Merlini 3 , Robert J. Greenberg 3 .1 Manchester Vision Regeneration (MVR) Lab, Manchester RoyalEye Hospital, Manchester, United Kingdom; 2 Manchester AcademicHealth Science Centre and Centre for Ophthalmology and VisionResearch, Institute of Human Development, University ofManchester, Manchester, United Kingdom; 3 Second Sight MedicalProducts, Inc, Sylmar, CA; 4 CHNO des Quinze-Vingts, INSERM-DHOS CIC 503, Paris, France; 5 Institut de la Vision, CNRS,UMR_7210, Paris, France; 6 Moorfields Eye Hospital, Moorfields EyeHospital, London, United Kingdom.Purpose: 1)To investigate whether Argus II subjects can locatehuman faces with their systems using a facial detection algorithm and2) whether detection speed improves when field of view that ismapped onto the Argus II implant is changed (i.e. demagnified).Methods: To date, more than 50 patients blinded by outer retinaldystrophies received an Argus epi-retinal prosthesis (Second Sight,Sylmar, CA). In normal use, a tiny video camera mounted on a pairof glasses gathers visual information. The video is subsampled tomatch the field of view of the implanted array and processed into 60pixels that characterize the average brightness of the scene at eachelectrode location.In the current study, the image of the scene acquired by the videocamera was processed using a face detection algorithm, resulting in avisual percept only where a human face was detected by theprocessor.A printed image of a face at normal size was place at random locationon a wall at a distance of 3 meters. A distractor image with equivalentsize and brightness was also placed on the wall at the same height.The subject was required to search for the face. In some trials, theimage processing algorithm captured a field-of-view that matched thefield-of-view of the implanted array (20 degrees diagonally) while insome trials the entire field-of-view of the camera (53 degrees) was©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Nanotechnology and Regenerative Medicinecaptured and “zoomed out” to fit the array. In a second experimentthe blind subject was engaged in a conversation with a sightedperson, who either faced the subject or turned away at some pointduring the conversation. The blind subject reported whenever he wasunable to detect the location of the face.Results: Five patients implanted with the Argus II System were ableto find the face 100% of the time with both magnifications. The timeto find the target was significantly shorter when using the wider fieldof-view.In the “real conversation” task, the blind subject was able torecognize within a few seconds when the other person turned away.Conclusions: Face detection in real world, i.e. at 2-3 m distance is achallenging task with a retinal implant. Using a device that takesadvantage of external image processing, we can provide facedetection functionality to blind patients. This feasibility studydemonstrated that image processing algorithms can enable patients toperform daily tasks that are not limited by the resolution or thesensitivity of the array.pixels. Each pixel has a photodiode, a differential amplification andan electrode for charge transfer to the adjacent retinal layers.Brightness gain and contrast can be adjusted by the patient manuallyvia two knobs on the external handheld unit.By means of an electroretinographical setup (Espion, DiagnosysLLC, Cambridge, UK) light responses from chips were measured onthe study eye in various gain-contrast settings in a special protocol of9 steps while raising stimulus intensities from 0.1 photopic cd/m2 to1000 photopic cd/m2 by 0.5 log steps.Results: The typical voltage versus log light intensity responsefunction of the subretinal implant for each contrast sensitivity andgain combination has a sigmoidal shape. Variation of the chipsensitivity shifts the curve left or right and allows to define theoptimal adjustment of all 1500 amplifiers in the chip to a givenluminance range of the ambient lightening. Variation of the outputgain changes the maximal output of the chip in the plateau of thecurve in order to adjust the point of saturation. Variations amongimplants are relatively minor.Conclusions: The function of subretinal visual microelectronicimplants can be measured objectively by special protocols usingstandard electroretinographical equipment. The exact knowledgeabout implant transfer characteristics is necessary for optimalparameter setting of artificial vision devices, especially in the trainingperiod after implantation as well as for objective measure of the chipfunction postoperatively. Suboptimal results in perception contrastand saturation can be avoided by assessing this curve in situ andusing of appropriate gain-contrast settings.Commercial Relationships: Paulo E. Stanga, OPTOS PLC (F),OPTOS PLC (C), OPTOS PLC (R), TOPCON CORP (F), TOPCONCORP (C), TOPCON CORP (R), SECOND SIGHT (F), SECONDSIGHT (R); Jose A. Sahel, UPMC/Essilor (P), Second Sight (F);Saddek Mohand-Said, None; Lyndon daCruz, Second SightMedical products Inc. (R); Avi Caspi, Second Sight MedicalProducts, Inc. (C), Second Sight Medical Products, Inc. (P);Francesco Merlini, Second Sight Medical Products, Inc. (P); RobertJ. Greenberg, Second Sight Medical Products, Inc. (I), Second SightMedical Products, Inc. (E), Second Sight Medical Products, Inc. (P),Second Sight Medical Products, Inc. (S)Clinical Trial: NCT00407602Program Number: 1767Presentation Time: 12:30 PM - 12:45 PMTransfer characteristics of electronic subretinal implantsmeasured by electrically evoked corneal potentialsKatarina Stingl 1 , Karl-Ulrich Bartz-Schmidt 1 , Dorothea Besch 1 ,Angelika Braun 2 , Florian Gekeler 1 , Udo Greppmaier 2 , AndreasSchatz 1 , Eberhart Zrenner 1 . 1 Center for Opthalmology, University ofTuebingen, Tuebingen, Germany; 2 Retina Implant AG, Reutlingen,Germany.Purpose: Hereditary retinal diseases lead to a progressive loss ofphotoreceptors often causing blindness in the patients’ middle agewithout an available therapy. Microelectronic subretinal implants aredesigned to replace the function of the degenerated photoreceptors.Methods: In the monocentric part of our clinical trial the subretinalAlpha IMS microelectronic visual implants (Retina Implant AG,Reutlingen, Germany) had been implanted in 11 patients in end stageretinitis pigmentosa. The implant's core is a subretinal chip with 1500Commercial Relationships: Katarina Stingl, Retina Implant AG(F); Karl-Ulrich Bartz-Schmidt, Retina Implant (P); DorotheaBesch, None; Angelika Braun, Retina Implant AG (E); FlorianGekeler, Retina Implant AG (F), Okuvision GmbH (F), RetinaImplant AG (C), Retina Implant AG (P); Udo Greppmaier, RetinaImplant AG (E), Okuvision GmbH (E); Andreas Schatz, None;Eberhart Zrenner, Retina Implant AG (F), Retina Implant AG (I),Retina Implant AG (C), Retina Implant AG (P), QLT Inc (C),Servier, Paris (C), Steinbeis GmbH&CoKG, Stuttgart (I), SteinbeisGmbH&CoKG, Stuttgart (C), Neurotech, USA (C), Pfizer, USA (C)Support: Retina Implant AGClinical Trial: NCT01024803439 Ocular Nanoimaging, Nanobiosensors and NanodiagnosticsWednesday, May 08, 2013 11:00 AM-12:45 PMExhibit Hall Poster SessionProgram #/Board # Range: 4674-4704/B0243-B0273Organizing Section: Nanotechnology and Regenerative MedicineProgram Number: 4674 Poster Board Number: B0243Presentation Time: 11:00 AM - 12:45 PMFunctional rescue for a full year after gene therapy in a preclinicalmodel of retinitis pigmentosa©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Nanotechnology and Regenerative MedicineKatherine J. Wert 1, 2 , Richard J. Davis 1 , Javier Sancho-Pelluz 1 ,Chyuan-Sheng Lin 1 , Stephen H. Tsang 1, 3 . 1 Ophthalmology, ColumbiaUniversity, New York, NY; 2 Institute of Human Nutrition, ColumbiaUniversity, New York, NY; 3 Pathology & Cell Biology, ColumbiaUniversity, New York, NY.Purpose: Mutations within the phosphodiesterase 6 (PDE6) complexare the third most common cause of autosomal recessive retinitispigmentosa (RP). In a pre-clinical model of RP, thePde6α D670G /Pde6α D670G mouse, there is a progressive loss ofphotoreceptor cells and neuronal function over time, due to amissense mutation in the alpha subunit of PDE6. No studies havebeen reported for any gene therapy for phototransduction defects thatlasts beyond six months of age in the mouse. We hypothesized that asingle treatment with a gene therapy vector to increase PDE6α levelswould result in at least a full year of visual function in the pre-clinicalmodel of RP (rescue for over half of the mouse lifespan and twice aslong as previously reported studies).Methods: An AAV2/8(Y733F) gene therapy vector driven by thecell-type specific rhodopsin promoter was utilized to deliver Pde6α:AAV2/8(Y733F)-Rho-Pde6α. A single subretinal injection of thisgene therapy vector was delivered at post-natal day five in the preclinicalmodel of RP, and results were analyzed between 7-12 monthsof age.Results: A single subretinal injection of the AAV2/8(Y733F)-Rho-Pde6α gene therapy virus was able to provide photoreceptor survivaland visual function for over half of the mouse lifespan. As previouslyreported, the untreated Pde6α D670G /Pde6α D670G mutant mouse eyeslost all photoreceptor nuclei by five months of age and lost visualfunction by two months of age. However, histological analysesdisplayed photoreceptors present in the treatedPde6α D670G /Pde6α D670G mutant mouse eyes after seven months ofage, and electroretinography confirmed these results with visualfunction present in the treated eyes for at least ten months of age.Conclusions: Studies have shown that the efficacy of the AAV viraltransduction is confined to the location of the subretinal bleb.However, a single subretinal injection of this therapeutic agent is ableto provide long-term rescue for over half of the mouse lifespan. Ourreport provides evidence that this gene therapy agent will preventdisease progression in human patients with RP due to PDE6αdeficiency.Commercial Relationships: Katherine J. Wert, None; Richard J.Davis, None; Javier Sancho-Pelluz, None; Chyuan-Sheng Lin,None; Stephen H. Tsang, NoneSupport: FFB, RPB, R01EY018213, and 5T32EY013933-12Methods: HAp was coated on the surface of pure-titanium through anovel coating technique, aerosol deposition (AD) method. Thetopography of the coatings was investigated with scanning electronmicroscopy (SEM). The rabbit cornea fibroblast (RCFB) was used toinvestigate the cytocompability. Cell morphology was studied usingSEM, and cell proliferation was analyzed by MTT at different timepoint. Cell migration was studied by scratch wound assay. Theexpression of integrin β4 was studied by reverse transcriptionpolymerase chain reaction (RT-PCR) and flow cytometry. In vivotest, the samples were implanted into healthy rabbit cornea andfollowed up by anterior segment optical coherence tomography(AS-OCT) for 12 weeks.Results: The SEM micrographs of nHA coating presented grain-likeappearance, while the surface of the bare-Ti showed a rigid metallicsurface (Figure 1). The morphological images by SEM showed mostcells on Ti surface were slim, shuttle-shaped morphology, while cellson the nHA modified surfaces were well spread and showedelongated, flattened morphology. The outcomes of MTT displayedthat an obvious proliferation of cells on the two different surfaces.The scratch wound assay showed cells on the nHA surface sealedwounds faster than that of bare-Ti at 24 h (Figure 2). Also, the highlevel of integrin β4 expression was illustrated on the nHAp-coatedsurface by RT-PCR and flow cytometry. In the animal model, nHAp-Ti implants were stably retained in the rabbit cornea, however thecorneal stroma anterior to the implants became thinner in the control.Conclusions: Our findings indicated that the surface composition andtopography have a significant effect on the biocompatibility ofimplant. HA-Ti can improve the cell activity in vitro as well as thetissue integration in vivo.Program Number: 4675 Poster Board Number: B0244Presentation Time: 11:00 AM - 12:45 PMThe effect of nano-structural hydroxyapatite on thebiocompatibity of artificial cornea skirt material in vitro and invivoYing Dong 1, 2 , Liqiang Wang 2 , Jingxin Yang 3 , Yifei Huang 2 , FuzhaiCui 3 . 1 Department of Ophthalmology, First Hospital Affiliated toChinese PLA General Hospital, Beijing, China; 2 Department ofOphthalmology, Chinese PLA General Hospital, Beijing, China;3 Department of Material Science and Engineering, TsinghuaUniversity, Beijing, China.Purpose: Titanium framework keratoprosthesis (Ti-KPro), as asynthetic artificial cornea has provided an option to the severecorneal blindness, but the bioactive of skirt material remained to beimproved. The purpose of this study was to evaluate the effect of thenovel biomaterial, nano-structural hydroxyapatite coated titanium(nHAp-Ti) on the material-cellular interaction in vitro and to assessthe integration of nHAp-Ti in vivo.Commercial Relationships: Ying Dong, None; Liqiang Wang,None; Jingxin Yang, None; Yifei Huang, None; Fuzhai Cui, NoneSupport: Technology innovation fund of general hospital 10KMM32Program Number: 4676 Poster Board Number: B0245Presentation Time: 11:00 AM - 12:45 PMFORMULATION OF A BIOCOMPATIBLEPHOTOPOLYMERIZABLE GEL TO PREVENT THEOXIATIVE DAMAGE OF THE CRYSTALLINE LENS©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Nanotechnology and Regenerative MedicineTongalp H. Tezel 1, 2 , Qun Zeng 1 , Martin G. O'Toole 3 , Andrea S.Gobin 3 . 1 Ophthal & Vis Sciences, University of Louisville,Louisville, KY; 2 Anatomy and Neurobiology, University ofLouisville, Louisville, KY; 3 Bioengineering, University of Louisville,Louisville, KY.Purpose: To develop a biocompatible photopolymerizable gel to sealoff the crystalline lens against oxygen diffusion to avoid cataractformation after vitrectomy.Methods: A composite gel was formulated to accommodate 13preset criteria: (1) Viscoelasticity, for intraoperative injectabilitythrough a 26-gauge cannula, (2) Spreadability, (3) Smoothness, (4)Ability to polymerize in situ, (5) Optical clearance, (6) Cohesiveness,to remain adherent to the lens capsule, (7) Iso-osmolarity, (8)Biocompatibility, (9) Oxygen impermeability, (10) Refractive indexclose to lens (n=1.336), (11) Surface energy of >40 dyne/cm, to avoidprotein and cell adhesion, (12) Elastic modulus >40 N/m 2 to preservethe accommodative ability of the lens, and (13) Biodurability. Forthis purpose, polyethylene glycol diacrylate (PEG-DA, MW: 3350-6000 kDa) base material was enriched with various concentrations ofhyaluronic acid (10-12 mg/mL, HA) or hydroxypropylmethylcellulose (30-100 mg/mL, HPMC) prepared in different solvents(PBS, HEPES, or dI H 2 O). After adding ascorbic acid (1 mg/mL) ortrehalose to the composite material, it was polymerized usingdifferent photo-initiators (acetophenone, eosin Y system and Irgacure2959). Resultant biogels were assessed in their compliance to thepreset criteria.Results: Ninety different permutations of the ingredients were testedfor their conformity to the required criteria. The best match wasattained by mixing 100 mg/mL PEG (6000 Da) with 10 mg/mL ofHA (viscosity 5200 mP.sec) and photoinitiating under a green LEDsource with Irgacure 2959 that was dissolved at 0.1 g/mL in 70%EtOH and added to PEG-polymer solutions to complex with theacrylate groups on the PEG molecules in a 1.2/1 ratio. The resultantclear (265-800 nm) gels have perfect spreadability, leveling,coverage, durability and cohesiveness with a thermally stable (25-37‘C) refractive index of 1.33, surface energy of 66 dyne/cm, andelastic modulus of 41.4 N/m 2 . These iso-osmolar gels resist proteinand cell adhesion, and can reduce oxygen diffusion 34 times.Conclusions: Formulated biogels can limit oxygen diffusion tocrystalline lens. This technology can eliminate tedious head-downpositioning and cataract formation after vitrectomy surgery.Commercial Relationships: Tongalp H. Tezel, University ofLouisville (P); Qun Zeng, None; Martin G. O'Toole, None; AndreaS. Gobin, University of Louisville (P)Support: Supported in part by an unrestricted grant from Research toPrevent Blindness, Inc, NYC, NYProgram Number: 4677 Poster Board Number: B0246Presentation Time: 11:00 AM - 12:45 PMEfficacy of a novel bioengineered corneal stroma fabricated by anew biocompatible cross linker in corneal stromal disease modelTakeshi Soma 1 , Michiya Matsusaki 2 , Masahiro Matsumoto 2 ,Hiroharu Ajiro 2 , Motokazu Tsujikawa 1 , Ryuhei Hayashi 1 , YoshinoriOie 1 , Mitsuru Akashi 2 , Kohji Nishida 1 . 1 Department ofOphthalmology, Osaka University Graduate School of Medicine,Suita, Japan; 2 Department of Applied Chemistry, Osaka UniversityGraduate School of Graduate School of Engineering, Suita, Japan.Purpose: Corneal stromal diseases causing severe visual loss can betreated by allogeneic corneal transplantation. However, there remainsmany problems such as immune rejection and the shortage of donorcorneas. We investigated the efficacy of a novel bioengineeredcorneal stroma fabricated by a new biocompatible cross linker inrabbit corneal stromal disease model.Methods: To create rabbit corneal stromal opacity model, a circularhorizontal lamellar dissection (3.0-mm diameter) was created at onehalfcornea depth using femtosecond laser (FSL) and trypan blue wasinjected into the stroma (n=8). For treatment, corneal opacificationwas removed and corneal stromal pocket was created by a 5.0-mmcylindrical dissection (100-μm height) via FSL. In 4 rabbits,biocompatible cross linker and type 1 collagen were injected into thecorneal stromal pocket. In 4 control eyes, nothing was injected.Corneal stromal reconstruction was assessed and histologicalexamination was performed 1 week after injection.Results: The corneal stroma was successfully reconstructed withrestoration of transparency in the injected eyes. The average cornealthickness in the treated eyes at preoperative day and postoperativeday 1, 2, 3, 5 and 7 were 370, 295, 274, 294, 299 and 280-μm,respectively. Those in the control eyes were 357, 247, 240, 248, 264and 241-μm, respectively. The reconstructed corneas weresignificantly thicker than those in the control eyes on eachpostoperative day (p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Nanotechnology and Regenerative Medicinewas processed by a femto-second laser to increase the chargeinjection capability. The electrical stimulating pulses consisted ofcathodic-first biphasic pulses (duration, 0.5 msec; frequency, 20 Hz;interpulse delay, 0.5 ms; number of pulses, 20) and they were appliedsequentially to each electrode with a delay of 0.45 ms.Results: All electrodes were functioning at the end of surgery in bothdogs. One month after the surgery, the fundus photographs andfluorescein angiograms showed no retinal damage in dog 1. Cornealartifacts elicited by the electrode array were recorded indicating thatthe electrodes were functioning.Conclusions: Our results indicate that the 2nd generation STS retinalprosthesis is feasible and can be considered for clinical use.Commercial Relationships: Takashi Fujikado, Nidek (P);Motohiro Kamei, None; Hirokazu Sakaguchi, HOYA Corporation(R); Hiroyuki Kanda, Nidek Co., Ltd. (P); Takeshi Morimoto,None; Kentaro Nishida, None; Haruhiko Kishima, None;Tomoyuki Maruo, None; Koji Osawa, NIDEK (E); MotokiOzawa, Nidek Co., Ltd. (E)Support: Health Sciences Research Grants (H24-medical device-004),the Strategic Research Program for Brain Sciences from theMinistry of Education,Culture, Sports, Science and Technology,Japan.Program Number: 4679 Poster Board Number: B0248Presentation Time: 11:00 AM - 12:45 PMAir transport system of oral mucosal tissue and cell sheets forclinical trialYoshinori Oie 1 , Satoru Andojo 1 , Natsumi Konno 1 , HiroshiTakayanagi 2 , Takayuki Nozaki 3 , Shizu Takeda 3 , Takeshi Soma 1 ,Motokazu Tsujikawa 1 , Kohji Nishida 1 . 1 Ophthalmology, OsakaUniversity, Suita, Japan; 2 Translational Research Center, TohokuUniveristy, Sendai, Japan; 3 Central Research Laboratory, Hitachi,Tokyo, Japan.Purpose: To develop air transport system of oral mucosal tissue andcell sheets for clinical trial using autologous tissue-engineered oralmucosal epithelial cell sheet.Methods: We developed a transport container that can maintain andmonitor the inside temperature and air pressure. Rabbit oral mucosaltissue was transported from Osaka University to Tohoku Universitywith this container. Epithelial cell sheet was fabricated with thetransported tissue, and was transported from Tohoku University toOsaka University. The tissue and cell sheets were evaluated beforeand 12 hours after transportation. Histological andimmunohistochemical analyses were performed. Cell viability andcell purity were evaluated by flow cytometry.Results: Temperature inside the container was kept 7 to 10 degreesCelsius for tissue, and 32 to 36 degrees Celsius for cell sheets. Airpressure was kept 990 to 1015 hPa during transportation. Basal cellsin the tissue were positive for p63. And cell sheets were wellstratified and harvested successfully after transport. The expressionpatterns of keratin 3/76, p63, and ZO-1were equivalent before andafter transport. The cell viability was 72.6% before transport and73.1% after transport. Epithelial purity was 82.0% before transportand 89.9% after transport.Conclusions: OralOra mucosal tissue and epithelial cell sheets canbe transported using this new transport system with qualitymaintenance.Commercial Relationships: Yoshinori Oie, Santen (F), HOYA (F);Satoru Andojo, None; Natsumi Konno, None; HiroshiTakayanagi, None; Takayuki Nozaki, None; Shizu Takeda, None;Takeshi Soma, HOYA corporation (R), Santen Pharmaceutical Co.,Ltd (F), Otsuka Pharmaceutical Co., Ltd (F); Motokazu Tsujikawa,Shionogi & Co. (C), Daiichi Sankyo Co. (F), Daiichi Sankyo Co. (R),Santen Co. (R), AMO Co. (R); Kohji Nishida, Alcon (C), Alcon (F),HOYA (F), Senju (F), Pfizer (F), Santen (F), Osaka University (P)Support: Health Labour Sciences Research GrantProgram Number: 4680 Poster Board Number: B0249Presentation Time: 11:00 AM - 12:45 PMFullerenol Protects RPE cells from Oxidative-stress InducedPremature Senescence via Activation of SIRT1Guo-Tong Xu 1, 2 , Chunchun Zhuge 1 , Jing-Ying Xu 2, 3 , Jingfa Zhang 2, 3 ,Guoxu Xu 4 , Lin Chen 2, 3 , Xiaoqing Liu 2, 3 , Hua Xu 2, 3 , Lixia Lu 2, 3 ,Weiye Li 5 . 1 Laboratory of Clinical Visual Science, Institute of HealthSciences, Shanghai Institutes for Biological Sciences, ChineseAcademy of Sciences, Shanghai, China; 2 Ophthal, RegenerativieMed, Tongji Univ School of Medicine, Shanghai, China;3 Department of Regenerative Medicine and Stem Cell ResearchCenter, Tongji University School of Medicine, Shanghai, China;4 Department of Ophthalmology, Second Affiliated Hospital, SuzhouUniversity, Suzhou, China; 5 Department of Ophthalmology, DrexelUniversity College of Medicine, Philadelphia, PA.Purpose: This study was aimed to study the protection andmechanisms of fullerenol on RPE cell senescence.Methods: H2O2 pulse induced premature senescence in both aRPE-19 cell line and primary porcine RPE cells. Senescence-associated(SA) β-galactosidase staining, DNA damage analysis and cell cycleanalysis was carried out to determine the protective function offullerenol in RPE cell senescence. The redox status in aRPE-19 cellswith or without fullerenol treatment was appraised bydichlorofluorescein (DCF) staining and catalase assay. Thesenescence and DNA damage related proteins were determined bywestern blot and immunofluorescence. The sirtuins activities weremeasured by sirtuin activity assay kit.Results: Fullerenol protected the RPE cells from H2O2-inducedsenescence through the blockage of p53 activation. H2O2 exposuretriggered DNA damage signaling pathway including the acetylationof p53 at lysine 382. In coincidently, accumulated γ-H2Ax foci andphosphor-ATM were both increased in H2O2-induced senescencemodel. This reactive oxygen species induced DNA damage signalingpathway activation could be partially rescued by fullerenol treatment.Also, as an activator of SIRT1, fullerenol intervention finallyinhibited p53 acetylation at K382, decreased p53 and its downstreamtarget p21, which accumulated in senescence cells, in response toH2O2. The senescent protection of fullerenol could be neutralized bySIRT1 inhibitors. The well known SIRT1 activator, Resveratrol,showed the similar protection in senescence RPE cells as fullerenolConclusions: Fullerenol significantly attenuates RPE senescenceinduced by H2O2 through its anti-oxidant activity and promotesSIRT1 activity. SIRT1 mediated p53 deacetylation is pivotal infullerenol anti-senescence mechanisms. Thus, this study suggests©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Nanotechnology and Regenerative Medicinefullerenol as the anti-AMD molecular and indicates SIRT1 as thepotential therapeutic target of AMD treatment.Commercial Relationships: Guo-Tong Xu, None; ChunchunZhuge, None; Jing-Ying Xu, None; Jingfa Zhang, None; GuoxuXu, None; Lin Chen, None; Xiaoqing Liu, None; Hua Xu, None;Lixia Lu, None; Weiye Li, NoneProgram Number: 4681 Poster Board Number: B0250Presentation Time: 11:00 AM - 12:45 PMReconstruction of Conjunctival Epithelium with Goblet Cells byCollagen VitrigelHuifang Zhou 1, 2 , Qiongyu Guo 1 , Michael Grant 3 , Jennifer Elisseeff 1 .1 Translational Tissue Engineering Center, Wilmer Eye Institute andBiomedical Engineering, Baltimore, MD; 2 Ophthalmology, ShanghaiNinth People’s Hospital, Shanghai Jiaotong University School ofMedicine, Shanghai, China; 3 Oculoplastics Division, Ocular andOrbital Trauma Center, Wilmer Eye Institute, Johns Hopkins Schoolof Medicine, Baltimore, MD.Purpose: The goal of this study is to evaluate the potential suitabilityof collagen vitrigel (CV) membrane as a substrate for engineeringconjunctival epithelium with goblet cells.Methods: Conjunctival epithelial cells were isolated from rabbitconjunctiva and were cultured in DMEM/F12 containing 10% FBS, 5µg/mL human recombinant insulin, 0.05 mM hydrocortisone and 10ng/mL EGF. The collagen type I vitrigel membrane was prepared in6-well culture plates as previously described [1]. Conjunctival cellswere seeded onto the CV and were compared to identical cultures oneither tissue culture plastic (TCP) or a standard collagen membrane.The viability and morphology of conjunctival cells on the differentsubstrates were investigated. Antibodies, including MUC5AC,specific for goblet cells, CK19 for conjunctival epithelium cells, andCK3 for corneal epithelium cells, were used to characterize thecultured cells.Results: Conjunctival epithelial cells demonstrated a high colonyformingcapacity that decreased with culture. The CV was atransparent membrane with sufficient mechanical properties with theaverage thickness of about 50 µm. The conjunctival epithelial cellswere easy to adhere to the CV membrane, and exhibited highviability (Figure 1) and proliferated faster than control groups. Thecells on the CV maintained their phenotype, as shown in Figure 2with the presence of CK19-positive conjunctival epithelium cells andMUC5AC-positive goblet cells. No CK3-positive cornea epithelialcells were detected.Conclusions: The CV membrane is a suitable substrate for in vitroculture of conjunctival epithelial and goblet cells. The artificialconjunctival epithelium can be manipulated and sutured, and it iscurrently being studied in vivo in a rabbit model of conjunctivalinjury.Reference1. McIntosh Ambrose W, etc. (2009) J Biomed Mater Res B ApplBiomater 90:818-31.Figure 1. Conjunctival epithelial cells on the CV. (A) Immediatelyafter seeding. (B) Four hours after seeding, the cells begin to adhere.(C)Three days after seeding, the cells showed proliferation. (D) Fivedays after seeding, the cells showed good viability (live cells, green;dead cells, red). Scale bar: 100 µm.Figure 2. Expression of MUC5AC and CK19 in conjunctivalepithelial cells after 5 DIC on the CV membrane by immunostaining.A and B: MUC5AC (green). C and D: CK19 (green). Nuclei werestained with DAPI with blue color. Scale bar: 100 µm.Commercial Relationships: Huifang Zhou, None; Qiongyu Guo,None; Michael Grant, Stryker CMF (C), Synthes CMF (C);Jennifer Elisseeff, Collagen Vitrigel (P)Program Number: 4682 Poster Board Number: B0251Presentation Time: 11:00 AM - 12:45 PMOverall visual performance is well described by threeindependent measures in Argus® II subjectsThomas Z. Lauritzen 1 , Anirban Das 1, 2 , Jessy D. Dorn 1 , Robert J.Greenberg 1 . 1 Second Sight Med Products, Inc, Sylmar, CA; 2 Dept. ofPhysics, USC, Los Angeles, CA.Purpose: To determine whether the performance of Argus IIprosthesis users can be characterized by a limited set of visualfunction measures. Reducing the number of outcome measuresneeded to characterize performance would streamline the assessmentof device benefit.Methods: The Argus II Retinal Prosthesis System includes a 10 x 6electrode array implanted epiretinally, a tiny video camera mountedon a pair of glasses, and a small external computer that processes thevideo and determines the stimulation current of each electrode in realtime. As part of a clinical trial, many outcome measure tests wererepeatedly administered at consistent time points; performance wasmeasured with the System ON and OFF. Additional outcomemeasures were performed only once on each subject. Here wedetermined how 14 different performance measures of Argus userscorrelate, including their ability to locate white objects on a black©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Nanotechnology and Regenerative Medicinebackground, their ability to detect the direction of motion of anobject, their spatial visual acuity, their ability to sort laundry based ongray scale, their ability to follow a line on the floor, their ability tofind a door, and their ability to read printed letters. Most performancedata were taken from the two-year follow up visit. A few tasks wereonly administered once during research projects. We then performedtwo types of multivariate analysis, Principal Component Analysis andSparse Component Analysis, to study if the outcome measures can bereduced in dimensionality to three (locating objects, determiningdirection of motion and their spatial visual acuity) while adequatelycharacterizing subject performance.Results: We find that the three measures (locating objects,determining direction of motion, and spatial visual acuity) correlatestrongly with the other 11 measures. Both multivariate analysismeasures reveal large orthogonal components between ON and OFFand between the three performance tasks; further dimensionalityreduction results in significant loss of information.Conclusions: These results show that the overall performance ofsubjects is well described by the three outcome measures. Themultivariate analysis results show that a complete description ofsubject performance needs to entail all three tasks. This suggests anoverall paradigm for assessment of the performance of visualprosthesis users, which is significantly less burdensome than thoseemployed previously.Commercial Relationships: Thomas Z. Lauritzen, Second SightMedical Products (E); Anirban Das, None; Jessy D. Dorn, SecondSight Medical Products (E); Robert J. Greenberg, Second SightMedical Products, Inc. (I), Second Sight Medical Products, Inc. (E),Second Sight Medical Products, Inc. (P), Second Sight MedicalProducts, Inc. (S)Support: NIH-NEI (EY020778)Clinical Trial: NCT00407602Program Number: 4683 Poster Board Number: B0252Presentation Time: 11:00 AM - 12:45 PMExcimer Ablation of Collagen-Based Corneal SubstitutesSilvia Odorcic 1 , Christopher Noel 1 , Debbie Mitra 1 , David Priest 1 ,Sabrina Taylor 1 , May Griffith 2 , Rejean Munger 1 , W. Bruce Jackson 1 .1 The Ottawa Hospital, Ottawa, ON, Canada; 2 Linköping University,Linköping, Sweden.Purpose: Biosynthetic corneal hydrogels have already undergonesuccessful implantation in human patients with keratoconus and mayone day serve as useful alternatives to human donor tissue forsurgical and refractive procedures. For synthetic materials to bebiocompatible, they should undergo laser ablation in a consistent andreproducible manner. The purpose of our study was 1) to determinethe feasibility and consistency of excimer ablation of collagen-basedcorneal biomimics (hydrogels), 2) to compare the ablation rates ofhydrogels with different mechanical properties.Methods: Twelve samples of four different hydrogel formulations(10% recombinant porcine collagen) underwent identical excimerablations (125 µm PTK, 6.0 mm). All hydrogels were cross-linkedusing a carbodiimide (EDC), epoxy-based cross-linker (BDDGE) orcombination (hybrid) to enhance their mechanical properties.Hydrogel ablations were captured using a specialized high resolutioncamera (optical profilometer). Custom software was used to extractablation rates by analyzing silhouette images of hydrogelsundergoing ablation.Results: All hydrogels were amenable to excimer ablation. Ablationrate was plotted against total ablation time for all samples. Hydrogelscross-linked with EDC exhibited extremely consistent ablation rateswith minimal inter-sample variability during all time points relativeto total ablation time. Hybrid hydrogels (EDC+BDDGE) exhibitedmoderate ablation consistency between samples, while those crosslinkedwith BDDGE alone displayed the poorest consistency inablation rates. The ablation rate of hybrid hydrogels was almost twiceas fast as that of EDC cross-linked samples. Hybrid hydrogels alsohad the most optimized mechanical properties (highest tensilestrength and elasticity).Conclusions: Cross-linked biosynthetic hydrogels can undergoexcimer ablation and therefore fulfill one important requirement ofsynthetic corneal substitutes: biocompatibility. By tailoring thehydrogel’s mechanical properties through cross-linking, ablationrates can also be manipulated. Our hybrid hydrogels have the highesttensile strength and elasticity, as well as the fastest ablation rates ofall samples tested. Such properties contribute to enhanced suturabilityand post-implantation healing. In the future, corneal hydrogels maysupplement human donor corneas with tissue-engineered alternatives.Commercial Relationships: Silvia Odorcic, None; ChristopherNoel, None; Debbie Mitra, None; David Priest, None; SabrinaTaylor, Allergan Canada (C); May Griffith, Univ. of Ottawa -OHRI (P); Rejean Munger, None; W. Bruce Jackson, NoneProgram Number: 4684 Poster Board Number: B0253Presentation Time: 11:00 AM - 12:45 PMHuman retinal progenitor cells as a tool for retinal repair:establishing cell lines for clinical studyPetr Y. Baranov 1 , Gary Brooke 2 , Sara Patel 2 , Michael J. Young 1 ,John Sinden 2 . 1 Schepens Eye Research Institute, Boston, MA;2 ReNeuron Ltd., Guildford, United Kingdom.Purpose: Loss of photoreceptors due to retinitis pigmentosa, agerelatedmacular degeneration and other age, trauma and geneticrelatedretinal degenerative disorders leads to progressive loss ofvision, and in some cases complete blindness. The successfulapplication of allotransplantation of mouse photoreceptor precursors,isolated from developing retina or differentiated from pluripotent celllines demonstrates the feasibility of restoration of retinal functionthrough cell therapy. However, time- and resource-consumingprotocols for photoreceptor differentiation from pluripotent cells, aswell as the potential for tumor formation, currently limits thetranslational potential of this approach. An alternative strategy is toisolate mitotically active human retinal progenitor cells (hRPC) fromdeveloping retina and expand them in vitro to the quantities requiredfor both preclinical characterization and clinical application.Methods: We isolated hRPC from two separate sources (week 18fetal retinae), expanded them under defined serum-free conditions ina low-oxygen environment, and established two cell lines. Thephenotype of the hRPCs were profiled by flow cytometry, functionalproperties assessed by the calcium imaging and the ability todifferentiate into photoreceptors on polycaprolactone and on pigretinal explants ex vivo evaluated with immunocytochemistry. Inaddition to flow cytometry, the equivalence of cell lines wasdetermined by microarray analysis (Affymetrix).Results: We found that both lines are mitotically active (Ki67 andPCNA positive), express stemness/eye field (Klf4, Sox2, Recoverin,SSEA4, Otx2, Pax6) and photoreceptor-precursor specific (Nrl, Crx,CD73) markers and can form photoreceptors (ROM-1, rhodopsin,opsin red/green) both in vitro and ex vivo. We also have established apanel of positive (CD73, Sox2, Pax6, HLA-ABC) and negative(CD133, CD38) markers as release criteria.Conclusions: hRPCs can be isolated from the developing retina, withdifferent cell lines and different passages of these cell linesdemonstrating equivalence. This is an important prerequisite toclinical application of this technology.Commercial Relationships: Petr Y. Baranov, ReNeuron (F); GaryBrooke, ReNeuron (E); Sara Patel, ReNeuron Ltd (E), ReNeuron©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Nanotechnology and Regenerative MedicineLtd (I); Michael J. Young, ReNeuron (F); John Sinden, ReNeuronLimited (E), ReNeuron Limited (I)Program Number: 4685 Poster Board Number: B0254Presentation Time: 11:00 AM - 12:45 PMHybrid vitronectin-mimicking polycaprolactone scaffolds andhuman retinal progenitor cell compositesElodie Lawley 1, 2 , Petr Y. Baranov 1 , Michael J. Young 1 .1 Opthamology, Schepens Eye Research Institute, Massachusetts Eyeand Ear, an affiliate of Harvard Medical School, Boston, MA; 2 ExeterMedical School, Exeter, United Kingdom.Purpose: Many advances have been made in the development oftreatments for retinal degenerative diseases such as age-relatedmacular degeneration and retinitis pigmentosa. The irreversible lossof photoreceptors is common to both and currently, no restorativeclinical treatment exists. It has been shown that progenitor celltransplantation can rescue retinal structure and function. This celltype can be collected from the developing neural retina, greatlyexpanded in vitro, and delivered into the degenerative host as asingle-cell suspension or as composite graft. Previously, we havedescribed several polymer scaffolds for culture and transplantation ofretinal progenitor cells (RPC). This tissue engineering strategyincreases donor cell survival, integration and differentiation.However, it conveys poor adhesive properties limiting its use andtherefore requires additional surface modifications.Methods: The aim of this work was to study hybrid vitronectinmimickingPCL scaffolds and their effects on hRPC adhesion,proliferation, differentiation and integration. In this study, we useCorning Synthemax II-SC as the vitronectin-mimicking substratewhich mimics the natural cell environment. In turn, it also enhancesexpansion to create a clinically relevant number of cells fortransplantation and supports differentiation. The differentiation ofhRPC on the polymer was assessed by flow cytometry for maturephotoreceptor markers, and the effect of the scaffold strategy on thetransplantation outcome by the explant approach whereby compositegrafts were placed on top of the pig retina.Results: We were able to successfully incorporate a vitronectinmimickingadhesive oligopeptide (Synthemax II) into a PCL scaffoldand show that the incorporation into PCL leads to dose-dependentincrease of cell adhesion, required for efficient hRPC culture. Theoptimal dose of Synthemax II was 30ug/ml (adhesion increased from10% to 65%), which was equal to standard fibronectin-coating.Incorporation of the oligopeptide into PCL did not change theinhibitory effect of PCL on hRPC proliferation: the populationincrease was 1.3 fold after 72 hours in culture.Conclusions: Hybrid vitronectin-mimicking polycaprolactonepolymers can serve as scaffolds for hRPCs, and the composite can beused to evaluate the potency of cells to be used in transplantationstudies.Commercial Relationships: Elodie Lawley, None; Petr Y.Baranov, ReNeuron (F); Michael J. Young, ReNeuron (F)Program Number: 4686 Poster Board Number: B0255Presentation Time: 11:00 AM - 12:45 PMStem Cell and Encapsulated Drug Delivery to the Inner Retinausing a Fibrin Polymer Spray SystemHari Jayaram 1, 2 , Megan F. Jones 2 , Richard M. Day 3 , Phillippa B.Cottrill 2 , Karen Eastlake 2 , Silke Becker 2 , G Astrid Limb 2 . 1 NIHRBiomedical Research Centre for Ophthalmology, UCL Institute ofOphthalmology & Moorfields Eye Hospital NHS Foundation Trust,London, United Kingdom; 2 Ocular Biology & Therapeutics, UCLInsitute of Ophthalmology, London, United Kingdom; 3 AppliedBiomedical Engineering, University College London, London,United Kingdom.Purpose: A significant obstacle to the development of stem cellbased therapies for the inner retina is the achievement of an adequatedistribution of cells across the neural retina. Fibrin scaffolds havebeen shown to provide a permissive environment for the proliferationand differentiation of embryonic stem cells in culture andcommercially available fibrin polymer spray systems have also beenused to deliver mesenchymal stem cells to human subjects in woundhealing studies. This study aimed to investigate the feasibility ofadapting a fibrin polymer spray to distribute retinal stem cells andenzymes encapsulated in microcarriers to the inner retina of the largermammalian eye through a conventional vitreoretinal approach.Methods: Human Müller glia with stem cell characteristics (hMSCs)were initially cultured in fibrin scaffolds. The fibrinogen:thrombinratio was optimised in order to maximise the viability and survival ofhMSCs in culture (as determined by Ki67/Apoptosis staining), tominimise scaffold density and to promote scaffold degradation inculture. PLGA microcarriers containing Chondroitinase ABC(ChABC) were then prepared by thermally induced phase separation(TIPS) prior to surgery. Pars plana vitreolensectomy was performedon cadaveric pig eyes stabilised with a vacuum fixated pad.Following fluid/air exchange, GFP labelled hMSCs and ChABCTIPS microcarriers were delivered with a fibrin polymer spraysystem into the posterior segment via an enlarged vitrectomy port.Cell distribution was assessed by fluorescence and confocalmicroscopy after a brief period in culture.Results: The survival and viability of hMSCs was not adverselyaffected by culture within fibrin scaffolds. 1mg/ml fibrinogenresulted in a thin scaffold with almost complete degradation after oneweek in culture. Study of the ex-vivo surgical model showed thataerosol delivery of cells through a conventional vitrectomy portoffered coverage of a large surface area as shown by the distributionof labelled cells.Conclusions: The fibrin polymer spray system shows potential as adelivery tool for cell-based therapies both to the inner retina andother body cavities, due to its ability to provide coverage of a largesurface area and ability to incorporate synchronous drug delivery.The availability of fibrin products licensed for human applicationmay help streamline the translation of this model towards clinicaluse.Commercial Relationships: Hari Jayaram, None; Megan F.Jones, None; Richard M. Day, UCL Business (P); Phillippa B.Cottrill, None; Karen Eastlake, None; Silke Becker, None; GAstrid Limb, NoneSupport: UCL / Medical Research Council (UK) Centenary AwardProgram Number: 4687 Poster Board Number: B0256Presentation Time: 11:00 AM - 12:45 PMA synthetic carrier for cultured corneal endotheliumimplantationMark Daniell 1 , Karl D. Brown 1 , Berkay Ozcelik 4 , PenelopeMcKelvie 3 , Hong Zhang 1 , Keren Abberton 2 . 1 CERA, Melbourne,VIC, Australia; 2 The O'Brien Institute, Fitzroy, VIC, Australia;3 Anatomical Pathology, St. Vincent's Hospital, Melborne, VIC,Australia; 4 Chemical and Biomolecular Engineering, The Universityof Melbourne, Parkville, VIC, Australia.Purpose: PurposeA tissue engineered corneal endothelium could overcome potentialproblems associated with donor tissues. We have developed a thin(50µm) synthetic hydrogel film suitable for corneal endothelial cell(CEC) culture and implantation. An ovine model was used to developand evaluate the film in vitro and in vivo.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Nanotechnology and Regenerative MedicineMethods: MethodsSynthetic hydrogel films (SHF) with superior properties weredeveloped using a novel approach. Ovine CEC (OCEC) density invivo was determined by specular microscopy and compared to celldensity achieved on tissue culture plastic and the hydrogel film asdetermined using Computer Assisted Steriological Toolbox (Leica).Films were implanted into an ovine anterior chamber for 30d beforehistological evaluation.Results: OCEC density in vivo was determined to be 3150cells/mm2in 14 month old merino sheep (n=3). Cell density at 28d was 3150(SEM 459) on SHF, 2530 (SEM 526) on collagen I coated SHF and3770 (SEM 201) tissue culture plastic (n=4). The films had sufficienttensile strength for implantation and readily un-scrolled within theanterior chamber. Histology determined that PHF did not produceand inflammatory response in vivo (n=4).Conclusions: Hydrogel films suitable for implantation that supportCEC cultivation have been produced. A combination of a SFH with acultured CEC monolayer may be able to replace the donor tissuelenticule used in Descemet’s Stripping Automated EndothelialKeratoplasty.Commercial Relationships: Mark Daniell, None; Karl D. Brown,None; Berkay Ozcelik, None; Penelope McKelvie, None; HongZhang, None; Keren Abberton, NoneProgram Number: 4688 Poster Board Number: B0257Presentation Time: 11:00 AM - 12:45 PMVision Restoration in Mouse Models of RP using Light-Gated G-Protein Coupled ReceptorsBenjamin Gaub, Michael Berry, Joshua Levitz, John G. Flannery,Ehud Isacoff. UC Berkeley, Berkeley, CA.Purpose: Retinitis pigmentosa (RP), an inherited eye disease thatleads to blindness following progressive photoreceptor degeneration,affects 2 million people worldwide. Despite this significant impact onthe entire population, there is no FDA approved treatment for RP todate.Recent experiments suggest that virus-mediated expression of lightsensitive ion-channels can restore visual function in rd mice in-vitroand in-vivo. However, the sensitivity levels of these optogeneticactuators post major limitations to vision restoration. Theillumination threshold is well above physiological light conditionsand is potentially harmful to the retina. More worrying yet, the largerthe eye, the more distance light has to travel until it reaches theretina, leading to a significant dop in light intensity. Moving to higherorder animal models will require actuators that are more senisitive tolight.Methods: Realizing the significance of this problem, we sought todevise light-gated proteins that can utilize the intracellular G-proteinsof retinal ON-bipolar cells (ON-BC). Employing the cell’s ownamplification machinery, we reasoned, might lead to enhancedsensitivity.We chose to study both natural and engineered light-gated GPCRs. Inorder to mimic the ON-BC's native signalling cascade, we designedlight-gated metabotropic glutamate receptors (Li-mGluRs) usingtethered azobenzene-based photo-chromic ligands. We also exploredwhether ectopic expression of verterbrate rhodopsin in ON-BCswould depolarize cells in a light dependent fashion. We were able totarget expression of light-gated GPCRs to ON-BCs selectively usingAdeno Associated Viruses (AAVs) and cell specific promotorelements.Results: Currently, we are in the process of testing the ability of ourlight gated GPCRs to reanimate ON-BC signalling in rd-10 miceusing in-vitro (MEA) and in-vivo (visually guided behavior)techniques. Preliminary in-vitro results suggest that vertebraterhodopsin is up to 1,000x more light sensitive compared tochannelrhodopsin under the same experimental parameters.Conclusions: Light-gated GPCRs expressed upstream in thedegenerating retinal circuit have the potential to enhance lightsensitivity. Harnessing the cell's internal amplification machinerywill help to lower the light intensity needed to "drive" the optogeneticactuators used as visual prosthetics.Commercial Relationships: Benjamin Gaub, None; MichaelBerry, None; Joshua Levitz, None; John G. Flannery, None; EhudIsacoff, NoneSupport: Grant 3PN2EY018241-01S1 from National Eye InstituteIRG: ZEY1Program Number: 4689 Poster Board Number: B0258Presentation Time: 11:00 AM - 12:45 PMConnective Tissue Growth Factor Protein Expression by MüllerCells is Bipartite as a Function of Substrate StiffnessWilliam J. Foster 1, 2 , Joshua T. Davis 2 . 1 Ophthalmology, Weill-Cornell Med Coll, Houston, TX; 2 Physics, The University ofHouston, Houston, TX.Purpose: Müller cells have been previously found to change theirexpression of connective tissue growth factor (Ctgf) mRNA by morethan 100-fold when cultured on substrates of different elastic moduli.To better understand the mechanosensitivity of Müller cells, wequantified the expression of CTGF protein utilizing a SandwichELISA with streptavidin-biotin and utilized immunohistochemistry todetermine the localization of the mechanotransduction markersYAP/TAZ in Müller cells as a function of substrate stiffness.Methods: A conditionally immortalized mouse Müller cell line(ImM10) was cultured on polyacrylamide substrates with a calibratedYoung’s modulus of 500 Pa, 1000 Pa, and 5000 Pa with glass as acontrol. A uniform coating of laminin was cross-linked to thesubstrates. For the ELISA studies, Müller cells were cultured for to24 days and the media was collected, stored at -80C until analysis,and changed every 3 days. All substrates were studied in triplicate.For immunohistochemistry, Müller cells were cultured for either 15or 21 days, and stained with antibodies to YAP/TAZ, utilizing astandard protocol.Results: The ELISA demonstrated unexpected, bipartite behavior, asa function of substrate stiffness. Cells grown on substrates firmerthan 1000 Pa had a peak in their protein expression at 15 days whilesubstrates of 1000 Pa and less had a peak in their protein expressionat day 21. Both the differences between the different substrates and asa function of time were significant (p=0.006, p=0.022, respectively;two-way ANOVA). Immunohistochemistry at day 15 demonstratedYAP/TAZ localization to the cytoplasm only in cells grown on firmersubstrates while studies at day 21 demonstrated localization to thecytoplasm only in cells grown on softer substrates.Conclusions: There appear to be two regimes in stiffness thatinfluence CTGF protein production in Müller, 1000 Pa and under andover 1000 Pa. Consistent with this finding, cytoplasmic localizationof the mechanotransduction markers YAP/TAZ corresponded withincreased production of protein. It is interesting to note that thetransition point in stiffness occurs at the point at which the Müllercells are normally found in vivo (1000 Pa).Commercial Relationships: William J. Foster, None; Joshua T.Davis, NoneSupport: NEI/NIBIB K08EY017112, NEI P30EY007551, NationalAcademies Keck Futures Initiative GrantProgram Number: 4690 Poster Board Number: B0259Presentation Time: 11:00 AM - 12:45 PM©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Nanotechnology and Regenerative MedicineCorneal Stromal Bioequivalents Secreted on Patterned SilkSubstrata by Corneal Fibroblasts and Stem CellsJian Wu 1, 2 , Yiqin Du 3 , Jelena Rnjak-Kovacina 4 , David Kaplan 4 ,James L. Funderburgh 3 . 1 McGowan Institute for RegenerativeMedicine, University of Pittsburgh School of Medicine, Pittsburgh,PA; 2 Ophthalmology & Surgery, University of Pittsburgh School ofMedicine, Pittsburgh, PA; 3 Ophthalmology, University of PittsburghSchool of Medicine, Pittsburgh, PA; 4 Biomedical Engineering, TuftsUniversity, Medford, MA.Purpose: Engineering of corneal stromal tissue is an important goalin the development of a functional corneal bioequivalent. In thisstudy, we conducted a comparison study to assess the applicability ofhuman corneal stromal stem cells (hCSSCs) versus human cornealfibroblasts (hCFs) in the generation of human corneal stromal tissueemploying a combination of surface guidance and growth factorsupplementation.Methods: Surface patterned silk films were prepared by solutioncasting, followed by surface-derivatization with Arg-Gly-Asp(RGD)-peptide. hCSSCs (passage 4) isolated from limbal stroma andhCFs (passage 6) from central stroma were seeded on the silk filmand cultured in serum-free media supplemented with FGF2, TGFβ3,and ascorbate-2-phosphate. After 9 weeks of culture, extracellularmatrixes (ECM) deposited by hCSSCs and hCFs on the substrateswere evaluated by transmission electron microscopy, wholemountimmunohistochemistry, and Western blotting of culture media. Geneexpression was examined by quantitative RT-PCR (qPCR).Results: Immunohistochemistry demonstrated that hCSSCsdeposited fibrous ECM abundant in type-I collagen on patterned silkfilms with RGD surface modification. The hCSSC construct was90~100 μm in thickness and featured stratified multilayered collagenfibrillamellae with orthogonal orientation, morphologically close tothat of native human corneal stromal tissue. In contrast, ECMsecreted by hCFs was only 20~30 μm thick and contained lessabundant type-I collagen than that from hCSSCs. Like scar tissue ofhuman corneal stroma, ECM from hCFs lacked orderly organization.Gene expression profiles showed that hCSSCs up-regulated mRNAfor several tissue-specific markers of keratocytes (KERA, ALDH,B3GNT7, CHST6,) much more substantially than hCFs. Westernblotting revealed that hCSSCs secreted proteoglycans includinglumican and keratocan modified with keratan sulfate. In contrast,these cornea-specific ECM components were hardly detected in hCFsecretedECM.Conclusions: These observations demonstrated that hCSSCs couldbe a powerful tool to develop an artificial bio-equivalent of humancorneal stromal tissue by stem cell tissue engineering strategy. hCFswould not be an appropriate cell line for corneal repair andregeneration, and might rather be responsible for scar tissueformation in the human cornea.Commercial Relationships: Jian Wu, None; Yiqin Du, None;Jelena Rnjak-Kovacina, None; David Kaplan, None; James L.Funderburgh, NoneSupport: NIH Grants EY016415 (JLF), P30-EY008098, EY020856(DLK), and Louis Fox Center for Vision Restoration, Research toPrevent BlindnessProgram Number: 4691 Poster Board Number: B0260Presentation Time: 11:00 AM - 12:45 PMKinetics of apoptotic death and oxidative damage in the retina ofP23H-1 rats and the protective effects of nanoceriaLily L. Wong 1 , Sudipta Seal 3, 4 , James F. McGinnis 1, 2 .1 Ophthalmology, University of Oklahoma Health Sciences Center &Dean McGee Eye Institute, Oklahoma City, OK; 2 Cell Biology andOklahoma Center for Neuroscience, University of Oklahoma HealthSciences Center, Oklahoma City, OK; 3 Advanced MaterialsProcessing Analysis Center, University of Central Florida, Orlando,FL; 4 Mechanical Materials Aerospace Engineering, Nanoscience &Technology Center, University of Central Florida, Orlando, FL.Purpose: Chronic rise in reactive oxygen species (ROS) is ahallmark in many forms of retinal degeneration. Excess ROS causesoxidative damage and cell death. Using the P23H-1 rats, we showedthat a single intravitreal injection of nanoceria, catalytic scavengersof ROS, delayed rod cell degeneration. However, the cellularmechanisms of this protection are unclear. We seek to understandhow nanoceria affect the kinetics of apoptotic death and whethernanoceria reduce oxidative damage in the retinas of these animals.Methods: We injected 2 µl of either saline or nanoceria (CNP)intravitreally to each eye of P23H-1 transgenic rats at postnatal day(P) 15 or 21. Assessments of cell death and oxidative damage wereperformed at 3, 7, or 14 days post injection. We used the 8-Isoprostane EIA kit to quantify lipid autoxidation in retinal tissuesand anti 8-OHdG on retinal sections to measure oxidative DNAdamage. We quantified the number of apoptotic (TUNEL+) profilesin the outer nuclear layer of retinas using the ApopTag PlusFluorescein In Situ Apoptosis Detection kit.Results: We observed a proportional increase in 8-isoprostane inretinas from wildtype, hetero- and homozygous P23H-1 ratsaccording to the severity of retinal degeneration. When CNP wereinjected at P15 in heterozygous P23H-1 animals, we observed areduction of 8-isoprostane in these animals compared to salineinjected ones at P29. When we examined the kinetics of cell death inthe retinas of heterozygous P23H-1 rats from P18 to P44, we foundTUNEL+ profiles peaked at P18 and followed a gradual declinethereafter. Few TUNEL+ profiles were observed in wildtype retinalsections of similar ages. We saw a 60% reduction of TUNEL+profiles in P18 P23H-1 retinas when CNP were administered at P15.Conclusions: Our results showed that a single application of CNP inthe vitreous reduced oxidative damage in the retinas of these mutantrats. We demonstrated that CNP were effective in preventing rod celldeath when administered early in age. We conclude that reduction ofphotoreceptor cell death after CNP application is likely due toreduction of oxidative damage in the retinas of these animals.Therefore using CNP to lower oxidative damage in retinaldegenerative diseases is a promising strategy to extend the functionallife span of diseased photoreceptor cells.Commercial Relationships: Lily L. Wong, 7347987 US Patent (P),7727559 US Patent (P), 2006242541 Australia Patent (P), 1879570Europe Patent (P); Sudipta Seal, UCF (P); James F. McGinnis,NoneSupport: NIH: P30EY021725, R01EY02211-01; OCAST: HR11-004; An unrestricted fund from RPBProgram Number: 4692 Poster Board Number: B0261Presentation Time: 11:00 AM - 12:45 PMConversion of human amniotic mesenchymal cells to retinalneural-like cells in conditional mediumYanqing Zhang 1 , Huiyong Wang 2 , Jiang Qian 3 . 1 Eye and ENTHospital of Fudan University, Shanghai, China; 2 Shanghai Xi MeiAesthetic and Plastic Center, Shanghai, China; 3 Eye and ENTHospital of Fudan University, Shanghai, China.Purpose: Human amniotic membrane-derived mesenchymal cells(hAMCs) has been shown the potential of three germ differentiations,and may be expected to show immunologic tolerance. In this study,we try to optimize differentiation protocols for inducing andmaintaining directed differentiation and gain functionalcharacteristics of induced retinal neuron from hAMCs, with regard totheir morphology, survival, migration and differentiation potential in©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Nanotechnology and Regenerative Medicinevivo.Methods: hAMCs were ex vivo expanded from human amnionicmembrane (HAM), and CD73, CD90, CD105, CD44, CD166, andCD45 expression was assessed by flow cytometry. Their Mesodermaldifferentiation was measured by osteogenesis and adipogenesis.Normal PCR and real-time PCR analysis were used to assess OCT-4,NANOG, REX-1, SOX-2, SSEA-4 and vimentin expression inhAMCs. Induced hAMCs were grafted to ventriculus lateralis of ratsand observed. Nestin, Map2, Ngn2, and Visinin were detected intransplanted rats by immunohistochemistry andimmunocytochemistry.Results: hAMCs express typical mesenchymal marker (CD73,CD90, CD105), and displayed strong adipogenic and osteogenicdifferentiation potential. And we induced hAMCs conversion ofretinal neural like cells in conditional medium with 4-stage protocol.The induced cells can be labeled retinal neuron progenitor and maturecell markers (nestin, MAP2, ngn2 and visinin). These cells were thentransplanted into the Lateral ventricle of rats, which survived andmigrated in the ventricle of rats, and can be labeled with ngn2 andvisinin.Conclusions: hAMCs can be induced to retinal neuron-like cells; thismay be a promising cell resource in clinical treatment for retinaldisease.hAMCs can be highly efficiently induced to express neuron markerswith defined mediumRegulation of Krüppel-like Transcription Factor (KLF’s) FamilyMembers Promotes Potent Axon Regeneration in the Adult RatOptic NerveYan Wang 1 , Akintomide Apara 1 , Murray Blackmore 2 , Dale P.Brown 1 , Michelle E. LeBlanc 3 , Alyson E. Trillo 1 , Jeffrey L.Goldberg 1 . 1 Ophthalmology, Bascom Palmer Eye Institute, Miami,FL; 2 Biomedical Science, College of health science,MarquetteUniversity, Milwaukee, WI; 3 PIBS, University of Miami, Miami, FL.Purpose: RGC axons fail to regenerate after optic nerve injury. Inour previous study, we found that KLF family members regulateintrinsic axon growth ability of retinal ganglion cells (RGCs) duringdevelopment. In this study, we asked whether knocking downmultiple KLFs, which suppress axon growth ability, andoverexpressing KLF7, which promote axon growth ability, wouldenhance axon regeneration in adult rats after optic nerve injury.Methods: The expression of KLF-9, -13, -16 and PTEN in adult ratRGCs were knocked down by intravitreal injection of AAV2-shRNA,and the expression of KLF-7 was up-regulated by intravitrealinjection of AAV2-VP16-KLF7. Optic nerve crush was performedtwo weeks after virus injection. 14 days after optic nerve crush, fulllengthof optic nerves to the optic chiasm were dissected andprepared for cryosection. Retrograde labeling with Fluorogold andanterograde labeling with Cholera toxin B were used to measureRGC survival and axon regeneration, respectively.Results: Compared to controls, simultaneous application of AAV2-shRNA anti KLF-9, -13, -16 significantly increased RGC survival upto 50%. Similar RGC protection was observed in AAV2-shRNA-antiPTEN treated animals. The number of regenerating axons in allexperimental groups was significantly more than in controls. Moreaxon regeneration was observed after KLF9 knockdown. Largenumbers of regenerating axons crossed the optic chiasm, the majorityof which extended contralaterally while a few axons grewipsilaterally.Conclusions: Knocking down KLF-9, -13, -16 by shRNA andoverexpression of KLF7 in adult rat RGCs, increase RGC survivaland induce axon regeneration after optic nerve injury. Our findingsindicate the potential translational application of these treatments foroptic nerve traumatic injury or degenerative diseases.Commercial Relationships: Yan Wang, None; Akintomide Apara,None; Murray Blackmore, None; Dale P. Brown, None; MichelleE. LeBlanc, None; Alyson E. Trillo, None; Jeffrey L. Goldberg,NoneSupport: AHAF; NEI EY020913; DoD W81XWH-12-1-0254hAMCs can be highly efficiently induced to express retinal neuronmarkers in vivoCommercial Relationships: Yanqing Zhang, None; HuiyongWang, None; Jiang Qian, NoneProgram Number: 4693 Poster Board Number: B0262Presentation Time: 11:00 AM - 12:45 PMProgram Number: 4694 Poster Board Number: B0263Presentation Time: 11:00 AM - 12:45 PMPositive charged nanoemulsion enhanced cyclosporine penetrateinto cornea after ocular topical applicationJunjie Zhang, Liya Wang, Tianyang Zhou, Jijun He, Huiyun Xia,Hongmin Zhang. Dpt of Pharmaceutical Science, Henan EyeInstitute, Henan Eye Hospital, Zhengzhou, China.Purpose: Cyclosporine (CsA) is an immunosuppressive agent thathas shown a strong immunosuppressive effect in a variety of animalmodels of transplantation and experimental autoimmune uveitis anduveoretinitis after systemic adminstration. However, it has limitedocular therapeutic effects due to its poor permeability across ocularbiological barriers while topical application. The purpose of thisstudy is to develop positive charged nanoemulsion for enhancing thepermeability of CsA across ocular biological barriers to treat uveitisafter ocular topical application.Methods: A series of positive charged nanoemulsions wereprepared.. The size and zeta potential of the nanogels are studied byMalvin laser particle size analyzer. The release kinetics of CsA from©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Nanotechnology and Regenerative Medicinethe nanoemulsion were measured and calculated using bulkequilibriumreverse dialysis bag technique. Ocular irritation test wasperformed in Japan White rabbits. The ocular distribution andpharmacokinetics of the CsA levels in the cornea, sclera, conjunctiva,aqueous humor, vitreous and whole blood were assessed after ocularsingle dose and loading dose topical application, resptively.Results: Laser particle size analyzer measurement demonstrate thatthe sizes of the nanoemulsions are around 29-51 nm at roomtemperature, and the zeta potential are +15-31mV. The accumulativerelease of CsA from the nanoemulsions were more than 80% within 2hours. Ocular pharmacokinetics of the nanogels strongly depend onthe and charge characteristics of the nanoemulsions.Conclusions: These findings indicate that positive chargenanoemulsion may be a promising vehicle for topical use of CSA totreat ocular immune-mediated diseases.Commercial Relationships: Junjie Zhang, None; Liya Wang,None; Tianyang Zhou, None; Jijun He, None; Huiyun Xia, None;Hongmin Zhang, NoneSupport: HNST Grant073108Program Number: 4695 Poster Board Number: B0264Presentation Time: 11:00 AM - 12:45 PMInduction of cytokeratin 3 expression in immortalized humanoral mucosal epithelial cells by the transduction of Pax6Yuzuru Sasamoto, Ryuhei Hayashi, Motokazu Tsujikawa, KohjiNishida. Ophthalomology, Osaka University Medical School, Suita,Japan.Purpose: To induce the expression of corneal epithelium-specificcytokeratin 3 (K3) in immortalized human oral mucosal epithelialcells (OKF6/TERT cells) using lentiviral transduction of Pax6.Methods: OKF6/TERT cells were transduced with two types oflentiviruses, each carrying one of the two variants of Pax6 (Pax6variant 1 and 2). The cells were cultured in modified keratinocyteserum-free medium (K-sfm) for 3 days after transduction and werecultured in keratinocyte conditioned medium (KCM) with 3T3 feedercells for another 11 days to stratify them. The gene expressions wereexamined with quantitative reverse transcription PCR (qRT-PCR)and immunofluorescence imaging on day 3 and day 14.Results: OKF6/TERT cells had no expression of K3 withouttransduction of Pax6 (Gene expression compared to GAPDH was0.01±0.00). OKF6/TERT cells expressed K3 on day 3 only whenthey were transduced with Pax6 variant 1 (Gene expression comparedto GAPDH was 7.31±0.66, P

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Nanotechnology and Regenerative Medicinewith murine induced pluripotent stem (MiPS) cells, which weredifferentiated for defined amounts of time and characterized withSEM and immunohistochemistry.Results: Scaffold physical features such as pore size and spacingwere found to significantly influence the growth and differentiationof iPSCs. Similarly the presence of nanostructure, introduced vialyotropic liquid crystalline templating, improved the diffusionproperties of the material and thus also positively influenced iPSCbehavior. The optimized materials produced were shown to supportthe differentiation of induced pluripotent stem cells toward matureretinal cell phenotypes.Conclusions: This work shows that physical properties ofphotopolymers can be successfully manipulated to meet the needs ofphotoreceptor regeneration applications. An optimized material ofthis kind; one that is biocompatible, implantable, and able toencourage growth and differentiation of mature retinal cell types,could lead to the successful transplantation of replacement cells andultimately, restoration of retinal function in patients who suffer fromlate stage retinal degeneration.Commercial Relationships: Kristan Worthington, None; AliasgerK. Salem, None; Allan Guymon, None; Budd A. Tucker, NoneSupport: NIH New Innovator Award (DP2OD007483), FoundationFighting Blindness, NSF (CBET-0933450)Program Number: 4698 Poster Board Number: B0267Presentation Time: 11:00 AM - 12:45 PMDevelopment of a bioengineered 3D-model of human conjunctivaIsabel Arranz-Valsero 1, 2 , Laura Garcia-Posadas 1, 2 , Ana Fernández 3 ,Antonio López-García 1, 2 , F. Javier Iglesias 3 , Yolanda Diebold 1, 2 .1 Ocular Surface Group, IOBA-University of Valladolid, Valladolid,Spain; 2 Networking Research Center on Bioengineering,Biomaterials and Nanomedicine (CIBER-BBN), Valladolid, Spain;3 Human Tissue Bank, San Francisco Clinic Foundation, León, Spain.Purpose: To develop a 3D-model of human conjunctiva by tissueengineering, using fibrin-based matrices.Methods: A biocompatible matrix, human fibroblasts and epithelialcells from bulbar conjunctiva from cadaveric donors were used toconstruct this model. Fibrin matrices were prepared using humanplasma or cryoprecipitate, following a method described by ourgroup. Both matrices had the same fibrinogen concentration (1.5mg/ml). We have previously optimized epithelial cell culture fromthat source testing different culture procedures and media.Immunocytochemistry was done against E-cadherin, cytokeratins(CK) 7 and 19, MUC5AC, vimentin and ki67 antigens in order tocharacterize cell phenotypes (n=3). Fibroblasts were grown inside thefibrin matrices and epithelial cells above them. Hematoxilin/eosinwas used to characterize the constructs and AlamarBlueTM assaywas used to measure cell proliferation at 3 and 7 days (n=5).Results: Fibrin matrices made from either human plasma orcryoprecipitate allowed cell growth (fibroblasts inside the matrix andepithelial cells above it). Human serum-supplemented medium wasthe best for epithelial cell culture, and it was chosen to maintain cellseededmatrices up to 14 days. Fibroblasts showed positive reactivityto the stromal marker vimentin, but negative to epithelial markers.Epithelial cells exhibited positive reactivity to E-cadherin, CK7 andCK19, but negative reactivity to vimentin. Some cells expressedMUC5AC. Fibroblasts inside plasma matrices showed betterproliferation rates than that of fibroblasts inside cryoprecipitatematrices (1.95 fold increase at 3 days; p = 0.004). On the contrary,epithelial cells showed higher proliferation rates when grown overcryoprecipitate-matrices (1.28 fold increase at 3 days; p = 0.022).Constructs seeded with both fibroblasts and epithelial cells, and madefrom plasma or cryoprecipitate, showed a 3.66 and 2.55 fold increase,respectively, in proliferation rates from day 3 to 7. 3D completeconstructs displayed similar consistency to that of humanconjunctival tissue.Conclusions: We have optimized a method to culture humanconjunctival cells from cadaveric tissue samples. In addition, wedemonstrated that fibrin-based matrices supported humanconjunctival cells growth when using them either as a scaffold(fibroblasts) or as a substrate (epithelial). It is then possible tobioengineer a fibrin-based 3D model of human conjunctiva.Commercial Relationships: Isabel Arranz-Valsero, None; LauraGarcia-Posadas, None; Ana Fernández, None; Antonio López-García, None; F. Javier Iglesias, None; Yolanda Diebold, NoneSupport: FEDER-CICYT Grants MAT2010-20452-CO3-01/03 andFPI Scholarship Program BES-2011-046381 (Ministry of Scienceand Innovation, Spain). Regional JCyL scholarship program andGrant VA132A11-2Program Number: 4699 Poster Board Number: B0268Presentation Time: 11:00 AM - 12:45 PMHybrid interphotoreceptor matrix - poly(caprolactone) scaffoldsfor human retinal progenitor cell culture and differentiationMichael J. Young 1 , Andrew Michaelson 2 , Petr Y. Baranov 1 , RebeccaL. Carrier 2 . 1 Schepens Eye Research Inst, Massachusetts Eye andEar, Harvard Medical School, Boston, MA; 2 Northeastern University,Boston, MA.Purpose: Stem cell therapy is a burgeoning technology fordegenerative disorders affecting organs with limited regenerativecapacity, such as the nervous system, including the retina. Althoughexperimental studies highlight the potential of stem cells orprecursors/progenitors delivered as a single-cell suspension, a tissueengineering strategy is a promising approach. Previously we utilizedfibronectin coating for poly(caprolactone) (PCL) scaffolds to inducephotoreceptor differentiation from human retinal progenitor cells(hRPCs). One alternative approach is the functionalization of thepolymer with extracellular matrix. Ideally, the incorporatedmolecules should provide adhesive and differentiation stimuli to thecells. Here we describe the differentiation of hRPCs on a hybridinterphotoreceptor matrix-PCL film.Methods: IPM was isolated from fresh bovine retinas, dissolved indichloromethane and mixed with 10% PCL. The scaffolds wereprepared using a previously described spinning method. hRPCs wereisolated from human fetal neural retina at 16 weeks of gestation andexpanded up to passage 9.The effect of IPM-PCL scaffold on hRPCmorphology was assessed by scanning electron microscopy anddifferentiation was assessed by flow cytometry analysis for maturephotoreceptor markers after 7 days in culture.Results: Lectin staining revealed that IPM lost its “honeycomb”structure during incorporation into PCL, although the adhesive abilityof the scaffold was greatly increased (70% vs 15%). However, wewere not able to achieve the rate of adhesion found for the currentstandard of fibronectin coating (95%). The rate of proliferation ofhRPCs on the IPM-PCL hybrid matrix was lower compared tofibronectin. Flow cytometry revealed the increased expression ofdeveloping photoreceptor transcription factors (Crx, Nrl) and maturephotoreceptor markers (Opsin Blue, Opsin Red/Green, Rhodopsin)after 7 days of differentiation on PCL-IPM, a result that wasequivalent to that obtained with fibronectin.Conclusions: The approach we describe allows one to overcomeboth the inherent lack of cell adhesion (specific for hydrophobicPCL) and the water-insolubility of IPM (which complicates its usagein vitro). We have shown that these hybrid scaffolds drive the hRPCfate towards photoreceptors, thereby providing a new tool in thedevelopment of retinal repair strategies.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Nanotechnology and Regenerative MedicineCommercial Relationships: Michael J. Young, ReNeuron (F);Andrew Michaelson, None; Petr Y. Baranov, ReNeuron (F);Rebecca L. Carrier, NoneSupport: NIH Grant EY021312-01Program Number: 4700 Poster Board Number: B0269Presentation Time: 11:00 AM - 12:45 PMDifferentiation Of Induced Pluripotent Stem Cells-DerivedNeural Crest Cells Into Corneal Keratocytes In VivoSatoru Yoshida 1 , Hideyuki Miyashita 1 , Miyuki Yasuda 1 , Emi Inagaki 1 ,Kazuo Tsubota 1 , Hideyuki Okano 2 , Shigeto Shimmura 1 .1 Ophthalmology, Keio University School of Medicine, Tokyo, Japan;2 Physiology, Keio University School of Medicine, Tokyo, Japan.Purpose: For application of induced pluripotent stem cells (iPSCs) toregenerativemedicine of corneal stroma, we show the ability ofiPSCs-derived neural crest cells (NCCs) to differentiate into cornealkeratocytes in vivo.Methods: For efficient differentiation of IPSCs into NCCs, weexamined modification of NSB method (Lee et al, 2010). To examinethe ability of iPS-NCCs to differentiate into corneal keratocytes, thecells were injected into mouse corneal stroma. The injected cellswere observed by confocal fluorescence microscopy. Cellmorphology was observed by phalloidin staining and the expressionof Keratocan, a corneal stroma specific keratan sulfate proteoglycan,was examined by immnohistochmistry using anti-ketatocanantibodies.Results: By induction with modified-NSB method, over 60% of cellswere isolated as NCCs using FACS. Four weeks after intrastromalinjection of iPS-NCCs, engraftment of the injected cells wasobserved. Phalloidin staining revealed dendritic morphology, whichis one of the characteristics of keratocytes. In addition,immnohistochmical analysis revealed the expression of Keratocan inthe engrafted cells.Conclusions: iPS-NCCs is useful cell source for regenerativemedicine of the corneal stroma by cell transplantation.Commercial Relationships: Satoru Yoshida, None; HideyukiMiyashita, None; Miyuki Yasuda, None; Emi Inagaki, None;Kazuo Tsubota, AcuFocus, Inc (C), Allergan (F), Bausch LombSurgical (C), Functional visual acuity meter (P), JiNS (P), Kissei (F),Kowa (F), Santen, Inc. (F), Otsuka (F), Pfizer (C), Thea (C), EchoDenki (P), Nidek (F), Ophtecs (F), Wakasa Seikatsu (F), CEPTCompany (P); Hideyuki Okano, Eisai Co.Ltd. (C), DAIICHISANKYO COMPANY (C); Shigeto Shimmura, NoneSupport: Highway program for realization of regenerative medicinefrom MEXT. The project for realization of regenerative medicinefrom MEXT.Program Number: 4701 Poster Board Number: B0270Presentation Time: 11:00 AM - 12:45 PMNanoparticle polyethylenimine-BMP7 Transfection Dose, AntifibroticEfficacy and Toxicity for the Rabbit CorneaChaitasi Naik 1 , Jason T. Rodier 1 , Ajay Sharma 1 , Audra Stallard 1 ,Ashish Tandon 1 , Alexander Klibanov 2 , Rajiv R. Mohan 1 . 1 Universityof Missouri - Columbia/Mason Eye Institue, Columbia, MO;2 Massachusetts Institute of Technology, Cambridge, MA.Purpose: The goals of study were to characterize polyethylenimine-DNA nanoparticles (NP) transfection dose to achieve high transgenedelivery at low toxicity in the cornea manipulating polyethyleniminenitrogen (N) and plasmid phosphate (P) molar ratio using an in vitromodel; and, test NP-BMP7 (bone morphogenic protein 7) genetherapy potency and safety to treat corneal scarring in vivo using arabbit model.Methods: New Zealand white rabbits and human corneal fibroblasts(HCF) generated from donor human cornea were used. NPpolyplexes with N/P molar ratio 4, 8, 15, 30 and 60 were preparedusing 22kD linear polyethylenimine and plasmid encoding GFP orBMP7 gene. Time- and dose-dependent transfection assays wereperformed for optimization studies. Corneal fibrosis (haze) in rabbitwas produced with excimer laser performing -9D photorefractivekeratectomy (PRK). NP-BMP7 was topically applied on rabbit eyewith custom technique for 5 minutes after PRK. Slitlampbiomicroscopy in live rabbits graded haze and ocular toxicity.Rabbits corneas were collected 4-weeks after PRK. Phase-contrastmicroscopy, trypan blue, MTT, TUNEL assays analyzed cellularmorphology, proliferation, viability and apoptosis death. Real-timePCR, western blotting, and immunfluorescence quantified genedelivery, efficacy and toxicity.Results: The NP-DNA gene transfer efficacy depended on N/P ratio.Higher N/P ratio in NP-DNA solution showed higher transgenedelivery (40-55%, p30 showed no-tomildphenotype, proliferation or viability loss in vitro (5-13%). Thetransfection with N/P 8 or 15 showed low toxicity. A single 5minutes topical NP-BMP7 (N/P 8) treatment showed significantlyless corneal haze (47-53%, p

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Nanotechnology and Regenerative Medicinerates were derived from an Alamar Blue (AbDserotec) assayperformed at timed intervals. For in vivo testing, 7-day old guineapigs were form deprived with diffusers for 4 weeks, beforeimplantation surgery involving sub-Tenon’s capsule injection of 100ul HyA hydrogel at the posterior pole of the eye. Ocular length wasmonitored for up to 2 weeks, when animals were euthanized and theeyes processed for histology, with Alcian Blue and Sirius Red asstains.Results: The modulus of the polymerized hydrogel was ~800Pa.After 14 days of culture, cells showed the highest proliferation rateon gels with the highest concentration of RGD (250uM), for whichthere was a 400% increase in cell number. In vivo, the implantedHyA hydrogel slowed ocular elongation and in one case, reducedocular length. Follow-up histology verified the hydrogel to be stillpresent 2 weeks post-surgery, as well as significant cell migrationand collagen deposition in the implant.Conclusions: The above data provide proof of principle for thepossibility of synthesizing injectible HyA hydrogels that arebiocompatible with scleral fibroblasts. Preliminary in vivo datasuggest that they can stabilize the sclera.Commercial Relationships: Mariana Garcia, None; Amit K. Jha,None; Kevin E. Healy, None; Christine F. Wildsoet, NoneSupport: Ezell Fellowship to MG, NIH Grant EY019628 to CFW &KEHProgram Number: 4703 Poster Board Number: B0272Presentation Time: 11:00 AM - 12:45 PMDevelopment of an Amniotic Membrane-Based Cell ScaffoldUsing Super Critical CO2 TechnologyDavid O. Zamora 1 , Jennifer L. Wehmeyer 2 , Heuy-Ching H. Wang 1 ,Robert J. Christy 2 , Anthony J. Johnson 1 . 1 Ocular Trauma, U.S. ArmyInstitute of Surgical Research, Ft. Sam Houston, TX; 2 ExtremityTrauma & Regenerative Medicine, U.S. Army Institute of SurgicalResearch, Ft. Sam Houston, TX.Purpose: Current ocular wound dressings on the market address suchbasic needs as wound protection, hydration, and healing. In additionto these criteria, there is a tremendous need for a dressing that is alsobiocompatible, can be used to deliver antibiotics for infection controlor growth factors for healing, as well as deliver cells for therapeutichealing. Commercially available human amniotic membrane (AM)meets some of these criteria, but current techniques for processingconfer undesirable tissue architecture and contain chemicalpreservatives that can decrease healing activity. We hypothesize thatsupercritical carbon dioxide (SCCO2) technology can be used as asingle step technique to process and sterilize AM tissues suitable tosupport the growth and potential delivery of therapeutic cells to theeye.Methods: Fresh AM was obtained from University Hospital, SanAntonio, TX (CRADA for Material Transfer #W81XWH-11-0441).The AM was processed for 10 minutes with 0.004% peracetic acidfor sterilization using a Nova 2200 supercritical carbon dioxidepressure chamber, and analyzed by routine histology. Human corneaepithelial cells (HCE), obtained originally from Dr. Ashok Kumar,were cultured (P8) using keratinocyte-serum free medium (GIBCO)supplemented with bovine pituitary extract and epidermal growthfactor. HCE were fluorescently labeled using 10mM CFSE and thenseeded onto the prepared AM for 0, 24, and 48 hrs and imaged usingfluorescent microscopy. MTT viability assays were also performedand proliferation rate determined at these time points.Results: SCCO2 treatment preserved the structural integrity of AM,while successfully sterilizing the tissue. HCE were successfullyseeded onto the surface of the AM and the tissue was able to supportmigration and proliferation of these cells, as determined byfluorescent microscopy and MTT viability assays.Conclusions: We have successfully combined existing SCCO2technology and the healing properties of AM to create an “off-theshelf,”sterile product for use in the clinic, as well as deployed withmilitary medics. This newly developed scaffold serves the dualpurpose of being a biocompatible, biodegradable bandage that canalso be used as a platform to deliver cells to the eye.Commercial Relationships: David O. Zamora, None; Jennifer L.Wehmeyer, None; Heuy-Ching H. Wang, None; Robert J.Christy, None; Anthony J. Johnson, NoneSupport: Core FundingProgram Number: 4704 Poster Board Number: B0273Presentation Time: 11:00 AM - 12:45 PMpRNA Nanoparticles for Intraviteal Delivery of siRNA in MouseRetinaDung V. Nguyen 1 , Sergio Caballero 1 , Sharon W. Matthews 2 , Jill W.Verlander 2 , Michael E. Boulton 3 , Peixuan Guo 4 , Maria B. Grant 1 .1 Pharmacology & Therapeutics, University of Florida, Gainesville,FL; 2 Medicine, University of Florida, Gainesville, FL; 3 Anatomy andCell Biology, University of Florida, Gainesville, FL; 4 PharmaceuticalSciences, University of Kentucky, Lexington, KY.Purpose: Considerable progress has been made in the deliverymethods of small interfering RNA (siRNA) through intravitrealinjection. However, naked siRNA lack the ability for targetingspecific cells. Phi 29 phage RNA (pRNA) DNA packaging motor canbe modified for nanotechnological applications. The domains allowfor the conjugation of different siRNA combinations onto ananoparticle. Additionally, one junction of the domain can be usedfor intracellular targeting for more efficient delivery, e.g., intoproliferating endothelial cells in neovascularization. We sought tocharacterize the properties of pRNA nanoparticles in the mouse retinaas an improved platform for siRNA delivery.Methods: AlexaFlour 647-labeled pRNA nanoparticles containingeither three- or four-way junctions were injected intravitreally intomouse eyes. Twenty four hours following injections mice wereeuthanized. Select eyes were fixed in 4% paraformaldehyde (PFA),the retinas dissected, and then sectioned for examination by laserconfocal microscopy. A separate proband of eyes were fixed forelectron microscopy (EM) by in vivo perfusion with 4% PFA inTyrode’s buffer and 0.25% glutaraldehyde. Retina sections fromthese eyes were incubated with primary mouse anti-AlexaFlour 647followed by gold conjugated anti-mouse antibody prior to EMcapture.Results: Imaging analyses revealed robust penetration of pRNAnanoparticles across retinal layers capable of reaching the retinalpigment epithelium (RPE) at 24 hours post-injection by bothdetection techniques. pRNA nanoparticles were observed in thechoroidal region, suggesting they can traverse the Bruch’s membrane.Nanoparticles conjugated to folate for intracellular uptake showed anassociation to cellular surfaces. No morphological abnormalities wereobserved at 24 hours post-injection. No differences in penetrationwere observed in the different pRNA nanoparticle formulations.Conclusions: pRNA nanoparticles can migrate efficiently toward theouter regions of the retina by 24 hours following intravitreal injectionand showed no toxicity based on morphology. Each formulation wetested with different structural properties crossed the retina makingpRNA nanoparticles useful as a platform for siRNA delivery. Withspecific cellular targeting, pRNA nanoparticles can potentiallydeliver combinations of therapeutics to sites of choroidal or preretinalneovascularization without compromising normal homeostaticprocesses.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group – Nanotechnology and Regenerative MedicineCommercial Relationships: Dung V. Nguyen, None; SergioCaballero, None; Sharon W. Matthews, None; Jill W. Verlander,None; Michael E. Boulton, None; Peixuan Guo, KylinTherapeutics, Inc (I), Biomotor and Nucleic Acids NanotechDevelopment, Ltd (I); Maria B. Grant, NoneSupport: Beckman Foundation©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.