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Research ArticleA Cornea Substitute Derived from Fish Scale:6-Month Followup on Rabbit Model

Fei Yuan,1 Liyan Wang,1 Chien-Chen Lin,2 Cheng-Hung Chou,2 and Lei Li3

Department of Ophthalmology, Zhongshan Hospital of Fudan University, Shanghai, China Department of Research and Development, Body Organ Biomedical Corporation, aipei, aiwan Department of Ophthalmology, Eye & EN Hospital of Fudan University, Shanghai, China

Correspondence should be addressed to Lei Li; [email protected]

Received March ; Accepted June ; Published June

Academic Editor: erri L. Young

Copyright Fei Yuan et al. Tis is an open access article distributed under the Creative Commons Attribution License, whichpermits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

A sh scale-derived cornea substitute (Biocornea) is proposed as an alternative or human donor corneal tissue. We adopt aregenerative medicine approach to design a primary alternative to the use o sh scale or restoring sight by corneal replacement.Biocornea with corneal multilayer arrangement collagen was implanted to rabbits by pocket implantation. Our study demonstratedthe saety and detailed morphologic and physiologic results rom the months o ollowup o rabbit model. In the peripheralBiocornea, the collagen brils were arranged in reticular ashion. Slit lamp examination showed that haze and an ulcer werenot observed in all groups at months postoperatively while all corneas with Biocornea were clear at both months and months postoperatively. Te interace o Biocornea and stromal tissue were lled successully and without observable immunecells at postoperative day . Moreover, the Biocornea was not dissolved and degenerated but remained transparent and showedno apparent ragmentation. Our study demonstrated that the Biocornea derived rom sh scale as a good substitute had highbiocompatibility and support unction afer a long-term evaluation. Tis revealed that the new approach o using Biocornea mayyield an ideal articial cornea substitute or long-term inlay placement.

1. Introduction

Te human cornea is the transparent outermost surace othe eye and major reractive element o the visual system;its unction depends upon its optical clarity. Blindness dueto corneal disease results both rom numerous degenerative

[], dystrophic [], inectious, and inammatory cornealdisorders and rom corneal damage secondary to ocularsurace disease. Te epidemiology o corneal blindness is

varied and complex, with inectious and nutritional cornealdiseases [], such as trachoma, onchocerciasis (river blind-ness), andvitamin A deciency (xerophthalmia), second onlyto cataract as a cause o blindness worldwide. Althoughcataracts are responsible in almost hal o the patients with

vision loss [], corneal damage and disease are the nextlargest cause []. Tese disorders are common in developingcountries in Asia and Arica and amenable to preventionthrough public health measures, whereas corneal scarring isthe most important cause o reversible blindness in children

[]. Corneal transplantation remains the main method orvisual rehabilitation once disease has affected corneal claritybut is dependent on the availability o corneal donor tissue,which is the major limiting actor in developing countries. Bycontrast, developed countries in the west have more inher-ited, degenerative, or iatrogenic disorders, such as Fuchs

corneal endothelial dystrophy [] and postcataract surgerycorneal decompensation, which have better prognoses.

Ulceration and trauma are responsible or an additional. to million new patients with corneal blindness annually[]. Te most successul and widely accepted treatment orcorneal blindness worldwide is ull-thickness replacement openetrating keratoplasty (PK). PK was the rst method operorming corneal transplantation and remains the mostcommon method. Approximately % o all transplants inAustralia are still perormed this way [].

Regardless o the technique, the undamental problemwith corneal replacement is a severe shortage o donortissue, resulting in approximately million [] untreated

Hindawi Publishing CorporationJournal of OphthalmologyVolume 2014, Article ID 914542, 6 pageshttp://dx.doi.org/10.1155/2014/914542
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Journal o Ophthalmology

patients worldwide. However, the gap between organ demandand donor availability has progressively widened, and thesevere shortage o organs or transplantation has resultedin the increasing use o expanded donor criteria, allowingthe inclusion o older donors as well as donors with milddisease. Tus, organ donation may involve the risk o the

transmittal o unwanted host actors, such as inectionsand malignancies []. Inectious microbes and unexpecteddiseases that are present in an organ donor have the potentialto be transmitted to the transplant recipient. Pretransplantscreening costs are high and will escalate as more rigoroustesting or an increasing number o transmissible pathogensis implemented [].

Recently, however, developments in bioengineered cor-neal substitutes designed to replace the ull or partial thick-ness o damaged or diseased corneas have been reported [].Tese range rom ully synthetic prostheses (e.g., keratopros-thesis) made rom polymethacrylates, that aim at replacingthe corneas reractive unction, to tissue-engineered cell-based constructs [] and hydrogels [] that also permitthe integration o the implant and regeneration o the hosttissues. Te most requent causes o corneal alterationsleading to keratoplasty are keratoconus, bacterial inections,poorhygienic contactlens wear, or trauma. Among microbialinections, bacterial inections are the most requent and aremainly complication. Some side effects o keratoplasty canbe inection (keratitis on the new transplanted cornea orendophthalmitis) [], transplant rejection, vision uctua-tion, glaucoma, and bleeding [], among others which areless reported. Teir use is thereore limited to cases in whichallogeneic tissue has ailed repeatedly or is contraindicated[]. A number o therapeutic strategies have been adoptedto treat donor deciency. Using human amniotic membraneas a biological substrate is a well-known technique or themanagement o ocularsurace reconstruction in patients withPK []. However, there are a ew problems with amnioticmembrane which still remain unresolved, such as sterilestorage or longer periods, the thinness o membrane thataffects the suture strength, wrinkling while transplanting,early degradation o the membrane, and the potential dangerothe spreado viruses [] and bacteria. Fishis a good sourceo collagen. Fish scales are composed o connective tissueprotein and collagen ( to %), covered with calcium salts(calcium phosphate and calcium carbonate) []. Tereore,sh scale may be an effective alternative source or collagenproduction [].

Our goal was to adopt a regenerative medicine approachto design a primary alternative to the use o sh scaleor restoring sight by corneal replacement. Specically, weproposed inducing regeneration o the damaged corneas byimplantation o an acellular and decalcied sh scale thatserves to acilitate regeneration by emulating the unctionso the highly natural extracellular matrix (ECM) scaffoldingo the cornea. Te substitute is cell-ree and o high bio-compatibility with host tissue to restore corneal unction,thereby avoiding the rejection reaction and the need orlong-term steroid use. van Essen et al. [] demonstratedsaety, biocompatibility, and regenerative potential o pocketimplantation o a sh scale in rat model. We report here the

saety, detailed morphologic, and physiologic results rom the-month ollowup o rabbit model. We specically evaluatedthe integration and stability o the implanted material andthe degree to which the implants enabled regeneration oendogenous epithelium.

2. Materials and Methods

.. Acellular and Decalcied Corneal Scaffold Preparation.Te tilapiash scales were cleaned in distilled water andcellu-lar components wereremoved usinga our-step detergent andenzymatic extraction process as developed by Lin et al. [].o increase pore sizes and porosity within the test samples,the acellular tissues were additionally treated with acetic acid.Te resultingdecellularized sh scales wererinsedextensivelyand stored. Decalcication o the material was perormed bybeing immersed in % nitric acid or hours at roomtemperature (R). Te materials were urther decalcied bybeing immersed in mL o solution A (% EDA, %

nitric acid) or - daysat

C with renewalo solution A dailydepending on the degree o mineralization o the scales. Aferdecalcication, samples were rinsed with % ethanol andstored in sterilized PBS at C or study. Finally, acellular anddecalcied sh scale-derived cornea substitute (8.0 0.8 mm[diameter],250 50 m [thickness]), consisting o collagentype I, was used or implantation.

.. Scanning Electron Microscopy. Scanning electronmicros-copy was used to examinethe surace morphology o acellularcorneal scaffold. Te acellular scaffolds were xed in .%glutaraldehyde in PBS (pH .) or min. Te xative wasthen aspirated. Afer being washed in PBS, scaffolds were

dehydrated in a graded series o ethanol solutions. Afercritical point drying (Quorum echnologies, model E,Guelph, Ontario, Canada), the samples were sputtered withgold using a SEM coating system (SPI, Sputter Coater,West Chester, PA, USA), and the probes were examined byscanning electron microscopy (JEOL, JSM-, okyo, Japan).

.. Histopathological Evaluation. Afer euthanizing the rab-bits with carbon dioxide, the isolated corneas were xed in% paraormaldehyde in . M phosphate buffer (pH .)or hours at room temperature or overnight at C. Afergradient dehydration in %, %, and % sucrose in . Mphosphate-buffered saline (PBS) each or hours, the cornea

was cut into halves and embedded at optimal cuttingtemperature compound (composed o glacial acetic acid,ethyl alcohol, and buffered ormalin). Sections were cut ona microtome (Leica RM; Leica Microsystems GmbH,Wetzlar, Germany) at um and stained with hematoxylinand eosin (HE) or histological examination. All eyes wereanalyzed or the organization o the epithelium, stroma, andendothelium and observed or inltration o immune cellsand ingrowth o corneal cells into the corneal scaffold. Teimmune inltrate was characterized based on morphology.

.. Surgical echnique. Sixteen New Zealand White rabbits,aged - months and weighing . kg, were used in the

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study. Animals were anaesthetized using an intramuscularinjection o ketamine hydrochloride ( mg/kg; Parnell Lab-oratories, Alexandria, Australia) and xylazine hydrochloride( mg/kg; roy Laboratories, Smitheld, Australia). One eyeo each rabbit was chosen at random or surgery. Tecontralateral eye o the implanted rabbit served as the

control. wo drops o tetracaine (Chauvin Pharmaceuticals,Kingston upon Tames, UK) were applied to the selectedeye. Following anesthesia, the rabbit eye to be implanted wasprolapsed anteriorly to improve surgical access. An anteriorlamellar dissection was perormed using a crescent blade.Te centre o the cornea was rst marked with a keratoplastytrephine (Solan; Xomed Surgical Products Inc., Jacksonville,FL, USA). A guarded diamond knie (Storz, Bausch andLomb, USA) was set to % corneal depth and a mmcircumerential incision was made to enable the ormationo a pocket incision and hence preservation o the cornealnerves. A depth o % has been previously shown to bethe optimal position or an implant or nutritional ow

(glucose and oxygen) in cornea stromal. A deep lamellardissection was then made, down to the posterior thirdo the cornea. A closed dissection technique was used bymaintaining the anterior lamellar ap in its original positionclose to the stromal bed and the crescent knie (Sharpoint,Angiotech, Vancouver, Canada) was sandwiched between the layers. With orceps gently holding the anterior ap inplace, the knie was gradually advanced perpendicular to thecornea curvature in a gentle sweeping motion, thus splittingthe cornea along a single lamellar plane. Sandwiching theblade between the stromal layers (and ensuring no tissuedistortion occurs) resulted in an even dissection, increasingthe likelihood o the dissection staying in the same tissue

plane throughout. A mm diameter implant was then oldedand inserted into the pocket. Pocket incisions have beenpreviously shown to maintain the nervous innervation othe cornea compared to a conventional LASIK like lamellarap. Te implantation was done sequentially starting withimplant. Following insertion, the implant was unolded usinga Paton spatula (Storz Instrument, Bausch & Lomb, USA)with a gentle sweeping motion. Te mm incision wasthen sutured with interrupted / nylon sutures. All rabbitsreceived the ollowing topical medications aferimplantation:neomycin-polymyxin B-dexamethasone ointment (Maxitrol;Alcon Laboratory, Inc., Fort Worth, X, USA) our timesper day, Pred Forte (Allergan; Irvine, Caliornia, USA) our

times per day, atropine % (Allergan; Irvine, Caliornia, USA)twice daily, and a lubricating viscous gel (Vidisic Gel; MannPharma, Berlin, Germany) once a day or week. Sutures wereremoved selectively in all rabbits at either or weeks aferimplantation.

.. Diagnosis of Corneal Recovery. External examinations oeach eye were done initially once daily or rst weeks andthen on a once-a-week basis over the entire course o thestudy. Detailed slit-lamp examinations o each rabbit wereperormed (in a double-masked manner) every other week.Eyes were examined or the presence o corneal peroration,

F : Te multilayer arrangement o collagen within Biocornea.

vascularization, or inection. Te diagnosis o corneal recov-ery was based on a subjective evaluation o corneal curvatureand stromal stability, including the presence o epitheliummigration by slit-lamp examination and uorescent staining.

3. Results and Discussion

Comparing to the native cornea, the D arrangement othe collagen similar to the corneal was studied. In thecentral Biocornea, the cutting edge consisted o multilayercollagen brils (Figure ). Te collagen layer wasorthogonallyarranged and was ormed by parallel bundles o brils,whereas the multilayer was a continuous plate o parallelbers. Te brils o the neighboring layers crossed eachother at an angle o about . In the peripheral Biocornea,the collagen brils were arranged in reticular ashion. Temechanical behavior o IOP resistant is directly affected bythe arrangement o the bers in its layer. Most pressurizedorgans andorganisms are reinorced by a crossed-helical berarray [], where bers are wrapped in lef- and right-handedhelices around the long axis o the structure []. Crossed-helical ber reinorcement permits constant pressure and

volume o eyeball easily [] and resisting occasional burst oIOP. Tese structures can stick easily because collagen bersare oriented parallel to the direction o the vertical orce [].Moreover, collagen multilayer structure also can maintain

water content to ca %.Te helical arrangement o bers makes sense or struc-

tures that are exible []. But cornea needs to remain inshape during daily scratch o eyelid, resisting external orcesthat could change the smoothness o surace and preventthe incision. For the rst days afer the injury, obviousconjunctival edema and eyelid swelling were observed oreach rabbit. By day , most o the eyes were covered withthe regenerating o pocket wound but small epithelial deectswere detected. From day on, the average areas o wounddeects were signicantly healing.

O the rabbits that underwent surgery, rabbit eyesreceived Biocornea and rabbit eyes as control group. Tere

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(a) (b)

(c) (d)

F : Five sutures were proceeded on the entrance o pocket with - silk and node buried inside; arrow indicated the transparencyimplant (a). Slit-lamp examination. Arrows indicate a successul inlay implant, which is almost imperceptible because o its extreme thinnessafer one week (b).Representativeslit-lamp micrographs o therabbit corneas.Cornea with Biocornea at months(c). Corneawith Biocorneaat months (d).

were our sham control eyes rom two rabbits, where thesurgery was perormed without implantation. One o therabbits with Biocornea and two rabbits as control developedmicrobial keratitis within the rst week o implantationand were excluded rom the study. A total o rabbitshad Biocornea and rabbits as control had successullyproceeded. One rabbit in the Biocornea group developedprogressive vascularization starting rom to weeks andhence was terminated earlier than originally planned. Teremaining rabbits with Biocornea were maintained to theoriginal predetermined time points without any problems.Tey also all revealed no immune response over the -monthperiod o study.

Slit-lamp examination showed that all control and sham-operated corneas were haze-ree at both and monthspostoperatively (Figures(b)and(c)). Haze and an ulcerwere not observed in all groups at months postoperatively(Figure (c)) while all corneas with Biocornea were clearat both months and months postoperatively. Te eyeswere enucleated at rst week, months, and monthspostoperatively. In the Biocornea group, a gross examinationo eyespecimensshowed that theinterace o Biocornea couldnot integrate perectly with the stroma and inltrate someimmune cells (Figure (a)) afer week. Biocornea remainedtransparent and well-rounded (Figure (a)). Te interace oBiocornea and stromal tissue were lled much better at

months postoperatively. Also, the transparency o Biocorneawas still very clear, and ew immune cells inltrates seem tohave obviously changed (Figure (b)). However, the integra-tion was lled successully and without observable immunecells at postoperative day (Figure (c)). Moreover, Bio-cornea was not dissolved and degenerated but remainedtransparent and showed no apparent ragmentation.

In vivo, biocompatibility o Biocornea depends on variousactors including the material itsel (permeable or nonperme-able polymer), the surgical procedure, the test animals, andthe postoperative conditions. O these actors, the surgicalprocedure plays an important role in determining the toler-ance o an inlay. In most mammals including humans, the

bundles enter the cornea at the periphery in a radial ashionparallel [] to the cornealsurace exerting important trophicinuences on the corneal epithelium and contributing to themaintenance o a healthy ocular surace.

4. Conclusions

Our study demonstrated that the Biocornea derived romsh scale as a good substitute had high biocompatibility andsupport unction afer a long-term ( days) evaluation.Tis revealed that the new approach o using Biocorneamay yield an ideal articial cornea substitute or long-terminlay placement. Tis new approach greatly decreased the

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(a)

(b)

(c)

F : Histology revealed the interace o Biocornea and stromal tissue. Asterisk indicates the location o Biocornea and arrow indicatedthe immune cells. Afer week implant, some immune cells inltrate around the Biocornea and exhibit some space in between tissue andimplant (a). Tere was signicant decrease o immune cells and tight junction o implant and tissue afer months (b). Te implant revealedhigh biocompatibility rom disappearance o immune cells and integration o tissue afer months (c).

biodegradation and improved its stability perormance as acornea inlay.

Conflict of Interests

Te authors declare that there is no conict o interestsregarding the publication o this paper.

References

[] L. Xu, Y. Wang, Y. Li et al., Causes o blindness and visualimpairment in urban and rural areas in Beijing: the Beijing eye

study,Ophthalmology, vol. , no. , pp. .e.e, .[] Q. Pan, X. Y. Li, and Y. S. Gu, Indications and outcomes o

penetrating keratoplasty in a tertiary hospital in the developingworld, Clinical and Experimental Ophthalmology, vol., no. ,pp. , .

[] D. . H. an, J. K. G. Dart, E. J. Holland, and S. Kinoshita,Corneal transplantation, Te Lancet, vol. , no. , pp., .

[] G. V. S. Murthy, P. Vashist, N. John, G. Pokharel, and L.B. Ellwein, Prevelence and causes o visual impairment andblindness in older adults in an area o India with a high cataractsurgical rate,Ophthalmic Epidemiology, vol. , no. , pp. , .

[] J. W. Streilein, M. R. Dana, and B. R. Ksander, Immunity caus-ing blindness: ve different paths to herpes stromal keratitis,Immunology oday, vol. , no. , pp. , .

[] N. G. Congdon, D. S. Friedman, and . Lietman, Importantcauses o visual impairment in the world oday, Te Journal ofthe American Medical Association, vol. , no. , pp. , .

[] S. Biswas, F. L. Munier, J. Yardley et al., Missense mutations inCOLA, the gene encoding the chain o type VIII collagen,cause two orms o corneal endothelial dystrophy, HumanMolecular Genetics, vol. , no. , pp. , .

[] J. P. Whitcher, M. Srinivasan, and M. P. Upadhyay, Cornealblindness: a global perspective, Bulletin of the World HealthOrganization , vol. , no. , pp. , .

[] K. A. Williams, M. A. White, P. R. Badenoch et al., Donorcornea procurement: six-year review o the role o the eye bankin South Australia, Australian and New Zealand Journal ofOphthalmology, vol. , no. , pp. , .

[] E. S. J. Kalter and . M. M. H. de By, issue bankingprogrammesin Europe, Te British Medical Bulletin, vol., no., pp. , .

[] . Eastlund, Inectious disease transmission through cell,tissue, and organ transplantation: reducing the risk throughdonor selection, Cell ransplantation, vol. , no. , pp. ,.

[] C. N. Kotton, Donor-derived inections in transplant patients,Clinical Microbiology Newsletter, vol. , no. , pp. , .

7/23/2019 914542

6/7


[] M. Alaminos, M. D. C. Sanchez-Quevedo, J. I. Munoz-Avila etal., Construction o a complete rabbit cornea substitute using abrin-agarose scaffold,Investigative Ophthalmology and VisualScience, vol. , no. , pp. , .

[] K.Nishida,M. Yamato,Y. Hayashida et al., Corneal reconstruc-tion with tissue-engineered cell sheets composed o autologousoral mucosal epithelium, Te New England Journal of Medicine,vol. , no. , pp. , .

[] M. Raat, F. Li, P. Fagerholm et al., PEG-stabilized car-bodiimide crosslinked collagen-chitosan hydrogels or cornealtissue engineering, Biomaterials, vol. , no. , pp. ,.

[] . Reinhard, A. Reis, D. Bohringer et al., Systemic mycophe-nolate moetil in comparison with systemic cyclosporin A inhigh-risk keratoplasty patients: years'results o a randomizedprospective clinical trial, Graefe's Archive for Clinical andExperimental Ophthalmology, vol. , no. , pp. , .

[] R. S. Ayyala, Penetrating keratoplasty and glaucoma, Survey ofOphthalmology, vol. , no. , pp. , .

[] J. M. Hackett, N. Lagali, K. Merrett et al., Biosynthetic corneal

implants or replacement o pathologic corneal tissue: peror-mance in a controlled rabbit alkali burn model, InvestigativeOphthalmology and Visual Science, vol. , no. , pp. ,.

[] S. Ferrari, V. Barbaro, E. di Iorio, A. Fasolo, and D. Ponzin,Advances in corneal surgery and cell therapy: challenges andperspectives or eye banks, Expert Review of Ophthalmology,vol. , no. , pp. , .

[] H. S. Dua, J. A. P. Gomes, A. J. King, and V. S. Maharajan, Teamniotic membrane in ophthalmology,Survey of Ophthalmol-ogy, vol. , no. , pp. , .

[] S. Krishnan, S. Sekar, M. F. Katheem, S. Krishnakumar, and. P.Sastry, Fish Scale Collagena novel material or corneal tissue

engineering,Articial Organs, vol. , no. , pp. , .[] F. Pati, B. Adhikari, and S. Dhara, Isolation and charac-

terization o sh scale collagen o higher thermal stability,Bioresource echnology, vol. , no. , pp. , .

[] . H. van Essen, C. C. Lin, H. J. Lai, A. K. Hussain, G. P. M.Luyten, and M. J. Jager, A biocornea o sh scalesrst resultso a research model,Acta Ophthalmologica, vol. , supplement, .

[] C. C. Lin, R. Ritch, S. M. Lin et al., A new sh scale-derivedscaffold or corneal regeneration, European Cellsand Materials,vol. , pp. , .

[] R. S. McCurley and W. M. Kier, Te unctional morphologyo starsh tube eet: the role o a crossed-ber helical array inmovement,Te Biological Bulletin, vol. , no. , pp. ,.

[] N. B. Ingels Jr., D.E. Hansen, G. . DaughtersII, E. B. Stinson, E.L. Alderman, and D. C. Miller, Relation between longitudinal,circumerential, and oblique shortening and torsional deor-mation in the lef ventricle o the transplanted human heart,Circulation Research, vol. , no. , pp. , .

[] B. L. Boyce, J. M. Grazier, R. E. Jones, and . D. Nguyen, Full-eld deormation o bovine cornea under constrained inationconditions,Biomaterials, vol. , no. , pp. , .

[] C. H. Lee, H. J. Shin, I. H. Cho et al., Nanober alignment anddirection o mechanical strain affect the ECM production ohuman ACL broblast,Biomaterials, vol. , no. , pp. , .

[] V. Ottani, M. Raspanti, and A. Ruggeri, Collagen structureand unctional implications,Micron, vol. , no. , pp. ,.

[] L. J. Muller, C. F. Marurt, F. Kruse, and. M. . ervo, Cornealnerves: structure, contents and unction, Experimental EyeResearch, vol. , no. , pp. , .

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