am 1 Nera ing 17 (2030) 055003 ‘apo 10 RHF AL STATA Journal of Neural Engineering First steps for the development of silk fibroin-based 3D biohybrid retina for age-related macular degeneration (AMD) ‘NehlaJemni-Damer™ ©, Atocha Guedan-Duran™, Jasmin Cichy, Paloma Lozano-Picaz', Daniel Gonzalez Nieto" ©, José Prex-Rigueio™""@,Francaco Rojo", Gustavo V. Guinea”, ‘Assunta Virtuoso! iil’ Michele Papa, Flix Armada Maresca", (Carlota Largo-Aran lador D Aznar-Cervantes' José L Ces and Fivos Panetsos"® + rer computing & Neue roots Resch Group Compete Universi of Madi Spin Inmostin Reach Group, neo Heth Rewarh Sn Cars Chal Hol SSC), Madi Spin 2 Thc autre cot eed ohare § Dp Hom! aging its Gy. Mati Macht 5 Ue St ni «Conte foc Biomedical ology, Pen Untwriy Mai, de Aso, Madi 7 omedial Rach NewoingCot in Boe, Botts od Some (CTDER BBN, Ma. Spin 5 Sit Boma Man Spin Dprtnnt of hss colgy nd Bioenpaoing ETSI elcmnstions PiU of Madd ps © Deparment Mn Senco de Cane Comal y Pte Roce Ueto Me spin © Dhron Hina aston” Neto Networs Morphology ah Beare Mca Ppaal Heh ad Peeve Mes, University of campus Vaneeli Naples Tay "2 Optnimalgy Seve, La Pre Uniesy Hosp ld, Spin 1 Experimenta urge: Unity onal La Pu (KSPAZ)- Me, Spa Deparment of Btehnolgy La Aer Miri) site Murcano denen y irl Agro y Ament (IMIDA, Een rovevemes Keyword sald, tinal pigment ethan, eras cle cl vi cla degeneration Abstract ‘Age-related macular degeneration isan incurable chronic neurodegenerative disease, causing, ‘progressive loss of the central vision and even blindness. Up-to-date therapeutic approaches ca ‘aly slow down he progression of the disease. Ojecive. Feasibility study for a multilayered, silk fibroin-based, 3D biohybrid retina. Approach. Fabrication of silk fibroin-based biofilms; culture of different types of cells: retinal pigment epithelium, retinal neurons, Mller and mesenchymal stem ‘ells; creation of a layered structure glued with silk fbroin hydrogel. Main results. In vitro evidence forthe feasibility of layered 3D biohybrid retinas primary culture neurons grow and develop neurites on silk fibroin biofilms, cither alone or in presence of other ces cultivated on the sume ‘biomaterial cell organization and cellular phenotypes are maintained in vitro forthe seven days of the experiment. Significance. 3D biobybrid retina can be built using silk sillavorm fibroin fils and Ibydrogelsto be used in cell replacement therapy for AMD aud similar retinal neurodegenerative diseases 1. Introduction Age-related macular degenerstion (AMD) is an incurable chronic neurodegencrtive disease, which ‘uses progressive loss of the central vision and even, blindness at its most advanced stage [= 4] Triggered by heterogencous, complex and still poorly understood pathogenetic mechanisms i is the main case of irreversible loss of vision among over 65 yo. subjects, and affects over 196 million people worldwide [1, 2, 4,5}. AMD acts on Broch ‘Rom IOP Angad ‘membrane, a 2-5 jm-thik layer that lies between the choriocapillary and the retinal pigment epithelium (RPE) (figure 1). ‘Under normal conditions this membrane (i) reg llstes the exchange of molecules, oxygen, muti ents and metabolic residues berween the retina and the choroid (i) together with the retinal ‘ment epithelium constitutes the external blood. retinal barrier and (ii) gives physical support for the correct adhesion and functioning of epithelium calls [5,7]. However, aging [8-0], oxidative stresstor asing 1 Neal ig 17202) 055008 ‘Carboy igure Left, Schematic epsentation of the uman ye with ait tacts Right. Magsfiation ofthe ma Nea Dames tal feces fected by age red nase degen in wch the dato of gerone in the ner ein te sede {Sachse thet pigment eit, Brac mcmrane andthe choad ca be breed [11-15] and several genetic factors [1616] cause the Bruch membrane to weaken, thicken and become ‘more rigid, which provoke & loss of permeability and other physiological dysfunctions [6]. Dysfunc- tions lead toa slowdown of the molecular exchange, and a consequent accumulation oflipids, cellular and ‘metabolic residues (originated in the retinal pigment epithelium and the surzounding tissue known also 2s ‘drusen’) which are accumulated between the basal ‘membrane ofthe retinal pigment epithelium and the Bruch membrane (19-22) All these changes cause a ‘chain of pathological events: los of the integrity of ‘theexternal blood-retinal barrier, death of retinal pig- ‘ment epithelium cells, then, death of photoreceptors and, st the end, permanent loss of the viewal function, Purthermore, the most aggressive stage of the disease (neovaseular/wet form) is characterize by the gener- ation of sub-choroidal neovessels that penetrate the ssubretinal space through the epithelium and thedam- aged Bruch membrane, and alter the 3D structure of the retina [22] Al current treatinents are aimed at relieving symptomsand at slowing dowa the progression of the dlisease 23]. Dueto the high prevalence and the wide socio-economic impact ofthe disease, as wells to the absence of clinical tretments and the growing aging ofthe world population, the development of effective ‘therapies for AMD isan imperative need fr which are collaborating biologie, doctors, chemicte/biochers istsand engineers [10, 24, 25]- Several research studies, suggest that the cre- tion and transplantation of artifical membranes ‘with RPE cells slow down the progression of AMD and even improve visual function [26-30]. Photore- ‘ceptor transplantation hae alzo shown good poten- tial as therapy for retinal degenerative pathologies with photoreceptor death, as in AMD advanced stages [25, 31-34]. Furthermore, with the great advances in differentiation of neural retinal cells from human stem cells, an unlimited number of photoreceptors can be obtained for transplantation purposes. However, most of the studies being conduc: ted with photoreceptors focus on injecting photoze ceptor precursors at the site of the injury of gen. cexsting SD retinal organoids in vitro [27, 3499}. ‘These last strictures have shown great potential for transplanting damaged cells, However, they have the disadvantage of their spheroid shape, which hinders synaptic integration and functional interaction with, the host. Despite all progresses, the great challenge cof AMD cell therapies isthe development of retinal implants, cither biological or biohybrid, capable to establish synaptic connectivity with the host tise ‘and restore viewal function [23, 25]. Furthermore, great effort is being made to develop biomimetic Bruch membranes with good adherence and survival properties for the cells to be transplanted and therefore capable to replace the damaged retinal tissue [29]. Researchers are working with both, eynthetic and astural materi als. The former include poly-methyl-methacryiate (PMMA) [40], modified polytetralluoroethylene (PTFE) [42], polyethylene terephthalate (PET) [42], poly-lactc-co-glycolic acid (PLGA) [43], poly-e caprolactone (PC) [44], etc, while the later include silk fbroin [5], collagen [46], hyaluronic acid (47) gelatin [45], ete. ell replacement therapies are a promising approach for AMD due to the surgical accessibil ity of the damaged tieue and the emall number of transplanted cells needed in comparison with other organs. The characteristic degeneration of retinal pigment epithelium and, in advanced stages of AMD, photoreceptors’ atrophy, has led to the development of cellular approaches aiming the replacement of these components [19]. RPE and photoreceptors have been transplanted both as a suspension and inte: rated into biomaterials. The first strategy has proven lower efficacy in terms of transplanted cell survival ‘nd integration [50] then biomaterial-encapsulated cells (51), which has led researchers to focus on the lattertor asing 1 Neal ig 17202) 055008 Researchers are working with biomster fale for RPE transplantation such as calls gen {52-50}, getin [5%, 60}, silk broin [61, 62] + polyethylene terephthalate (PET). (s3} pelylectic acid-co:glycolie acid) (PLGA). [o4, 15], ete. Several biomaterials have shown good biocompatibility and allowed the formation of an RPE polarized monolayer in vitro [1 65-09]. This IDE cells can be extracted from adult man RPE stem cells, RPE derived feom embryonic stem cells or from induced pluripotent stern calls (PSC), ete. Maintenance of RPE cell’ Functionality on the bio- snstrial is ruil the succee ofthe transplant and only some in vitro studies assessed it [70-72]. The highest RPE survival once transplanted was reported fora gelatin and silk ibroin/pelyeaprolactone/geltin scaffold reaching 3 months on porcine (73) snd rab bit eyes [7 eepectively Also noteworthy are the ‘ests of RPE implanted on a plasms modified poly- incubator. Then, enzyme activity was neat- zalized by dipping the eyeballs in DMEM-F12 + 109% FBS +196 P/S (M) medium. Eyes were then dissected along the comnea-sclera ‘edge and cornea, iris epithelium, and lens were gently removed. Retina and posterior sclera were put in ‘M and incubated for 25 min in presence of 37 °C 596 CO; to help the separation of the neural ret ina from the retinal pigment epithelium, After neural retina removal, the retinal pigment epithelium layer attached to the choroid was cut into a four-leaf shape structure, Epithelium sheet vas carefully scrapped ‘of fromthe inside to outside and the fragments on, tach flap were collected and transferred into a 1.5 ml Eppendorf tube prior to centrifugation at 1,000 rpm for 5 min at room temperature, The supernatant wos discharged, and retinal pigment epithelium cells were gently resuspended in a 1.0 ml complete medium (DMEM-FI2 + 108% FBS + 19 P/S). Cells from two rats eyes were put into one well of the 48-well plate ‘with 500 jl complete medium. Cells were incubated 3 Nea Dames tal in growth medium containing in pretence of 37 °C 59% COs, 2.2.4, Retna dissection, primury coll eulures ana neurons isolation Cal isolation performed according to the proced ture deseribed in Rosca et al, 2015 [105] with some modifications, Briefly retinas were dissected out from. whole eyeball according to the standard retina peel- off (a already explained in the previous section) and {uedted with 0.25% Trypsin-HIBSS solution (Sigma: Aldrich, St. Louis, CA, USA) for 10 min for singe cells, release. Single ces (5,000 cllswell) were seeded on. preconted (10 mg poly-L-lysine and 100 mg laminin) silk fibroin films in 48-well plates and cultured in Neurobasal media supplemented with 5% FBS and 1% P/S. Under a microscope cells putin wells with complete meditim were confirmed to be evenly die twibuted before transfering inta a 37 °C 5% CO, incubator, Culture dish was kept in undisturbed con. dition for at least 48 h to allow cell attachment to the dish, Culture medium was half-changed every 2 through removing haf of it and then by adding fresh culture medium. 2.24, Miller cll ine culture ‘Miller cell Line MU-PEIL cells were grown to con- fluence in growth medium containing DMEM- F12 + 10% FBS + 19 PYS in presence of 37 °C 5% (CO, incubstor. At easly passage (P6-P9), cells were tuypsinized, centrifuged at 1,000 spm for 7 min and seeded to thefilme, Before cell seeding, file were pre conditioned overnight in culture medium at 37 °C. Calls were left incubating undisturbed at 37 °C and 59% CO, for 48 b to prevent them from flowing out the film, Culture medium was changed every 2 days 2.2.5. Mesenchyynal call cultures Isolation and expansion of CD-1 mice bone marrow sevenchymal stem cells was performed as described in Martin-Martin et a, 2019, Scientific Reports). Cell, viability om sill fbroin films wae assayed Calccin AM. (cBioscience; Catt 65-0853) staining for 20-30 min at 37°C, Cells were imaged under fluorescence micro scope (Leica DM13000, Nussloch, Germany) coupled with » Laicn DECHOPK camera, Sprending aren and numberof processes in mesenchymal ellsgrowingon, tueated plastic (TCP) or sill fibrin films were eval ated with Image} software (NIH), 2.2.6, Attachment of ell 40 silk fibroin scaffolds Silk fibroin flms (figure 3) were sterilized and pre conditioned for cell culture by two siccessive rinses with ethanol 70° for 1.0 exch ina laminar low cabs inet, and then rinsed with ethanol 50° and 30° for 10 iin each, followed by a final tinge, thoroughly with sterile, deionized water. Then, they were pre conditioned overnight in culture medium at 37 °C, Aer that, they were adhered to culture well floorsoP Paising 1 Neal ig 17202) 055008 silk hydrogels ‘2 ‘Tension (Kpa) Nea Dames tal silk biofilms ear. Deformation (%) Figuse Top Appearance ofthe ld il nn hydrogel aa elo mast ina tube (A), an ofthe ik Sci bio in ‘Shr lite (8) Boon: Carve of esc modal ofthe ater (rope (C) shoe (D) sms deformation and their surfices were modified by overnight cost: ing with 10 mgpoly-L-Iysine + 100 mg laminin under UV light, to increase cells adhesion, Polymers were then rinsed three times with GBSS, Cultured cells ‘were dissociated into cell suspensions separately and 300 1 were seeded onto each scaifold. After cell seed- ling, mats were incubated at 37 °C for 30 min to allow cells attachment, medium was added to prevent drying, and cells were incubated for further 30 min, Finally, growth medium was added into the inserts in the wells and cultured in the humidified CO, incub- stor. Medi was changed every 2 days. 2.2.7. In vitro eration of biohybrid retinas ‘The implant of the biohybrid into the eye is not an easy process because (i) fragility does not allow biokybrids tobe picked up and introduced tothe eye tissue (i) inflammation provoked to the neural tis- sue by the surgical intervention must be controlled and (ii) to survive, cells in the biohybrid retina mast bbe surrounded by a neuroprotective environment. For these reasons we decided to wrup the biohybrid top with ail bvoin gel (gure 3) with mesenchymal stem cells and Miller cells encapsulated in it, two types of cells well known for their neuroprotective and neuroregenerative secretoms ‘The structure ofthe artific by attaching a biofilm with retinal pigment epithe liam cells to afl with neurons, thus allowing con tact between the two types of cells. In a cylindric (08.0 mm) mold we create a lier of silk fibroin hydrogel with mesenchymal stem cells and Mller cells; then we insert a film with retinal pigment ‘epithelium cells; we add a second film with nur: ‘ons; and we conclude by adding a final ayer of sll fbroin hydrogel. The hyerogel’mesenchymal stem, retina was created 6 cells mixture protects cells and Keeps the struc ‘ute together, provides nutrients and neuroprotective snd antiinflammatory molecules while isolating the implant from the hostile environment, thos favoring the survival of the implanted cells 228, Bualuation of cell ervival (Gnnrmohistochemistry/imomusnofluorescence) Alter 7 culturing, cells om films were washed with, PBS (0.1 M NaC, pH 7.2), fixed in cold 49 buf fered paraformaldehyde (PEA) (15 min), permeab ilized in 0.01.09% ‘Triton X-100 (15 min), washed with PBS (0.1 M NaCl, pH 7.2) and then incub- ated with primary antibodies overnight at 37 °C: anti-RPE-65 (RPE cells, anti-NeuN (neurons) and lial fibrillary acidic protein (anti-GPAP, Miller lia). Samples were then rinsed three times with PBS and reacted with species-specific immunoglobulin conjugated. The secondary antibodies were Cy3 AffiniPure Goat Anti-Rabbit IgG (H+ 1) (1:800) (111-165-003, Jackson ImmunoResearch) and Fluor escein (FITC) AffiniPure Goat Anti-Mouse Ig ( + L) (115-095-003, Jackson ImmunoResearch), incubated for 3h at room temperature. Sections were need three times with PBS and cell nuclei were phenylindole counterstained with 4, 6-diamidino (DAPI; 1.0 gml-!, Molecular Probes), 3. Results 43.1. Silkfibroin supporting structure Ten to twenty micrometer-thick sik fibroin films and fluorescein isothiocyanste-stained hydrogels igure 3) were used to build our biohybrids. Mech- nical tests (= 5 in both cases) showed very similar clastic moduli (13.44 + 1.67 and 14.90 + 1.88 MPa, respectively) suitable to be used for the same implantstor asing 1 Neal ig 17202) 055008 [65]. Maximum deformations were in the range of 10%-20% for both materials but with maximum tension of 1194.64 +: 191.55 kPa and 1.76 + 0.09 KPa for films and hydrogels, respectively. ‘We have previously described the excellent com: patibility of silk fibroin to support the growth of speciic cellular lineages (Martin-Martin ef al, 2019, Scientific Reports). Figure 4 ilustrates the ability of silk ibroin (8%) to favor the expansion and prolif ‘eration of mesenchymal stem cells, eel population with trong repercussion in regenerative medicine. In agreement with our previous study (Martin-Martin ef al, 2019, Scientific Reports) and independently of the substrate (TCP of silk fibroin) these eels ‘were perfectly able to attach and extend processes showing the typical spindle-shaped fibroblast like ‘morphology characteristic of this cell population, (igure 4) Silk tolerability has alsa been demonstrated in the context of macula and retina degeneration, In a couple of studies, no inflammatory response, neither eytotonicity was observed after in vivo trans: plantation of silk fibroin ecafolde or nanoparticles alter subretinal or intravitreal injection respectively [105, 107]. In both formats, this material suppoets the growth and function of RPE cell, Other formu- lations based on silk fibroin-tropeclastin composites, support the growth of RPE cells [108]. In an interest ing study. an advanced photovoltaics semiconductor~ ‘based organic prosthesis formulated with sill fibroin war implanted in the degenerated retina to pro- duce photo-stimulation of residual neural networks ‘promoting visual recovery in an animal model of ‘photoreceptor degeneration [109]. These in vitro and in vivo approaches provide the necessary framework. to push the fabrication of like-retna structures based on this material ‘32, Invitro building and testing of the biohybrid 4.2.1 Creation ofa biohybrid retina using cll cultures ‘and artificial substrates ‘We ereateda 3D retina structure with to silk fibroin biofilms attached each other, thus allowing seeded ‘neurons on one film to contact with the seeded ret- inal pigment epithelium celle in the other A mes ‘enchymal stem cells-enriched glued the two films together (Bure 5). Best results were obtained with ‘alms built with functionalized silk fibroin, which providesbinding sites for retinal cll figure 5) Func tonalization allows us to adapt the substrate for dif- ferent cell types. 4.2.2 Survival of in vito create biohybrd retinas Filins are not cjtotoxie and favor cell viabil- ity and adhesion (figure 4). Cells seeded on PLLilaminin-coated films show a higher adhesion rate than on not treated ones. The adequacy of the fabricated substrates is demonstrated by the survival 7 Nea Dames tal of the cells obtained by primary culture for up to 7 4 invitro (Bgures 5-8). Cultured retinal clls need a suitable substrate for their survival, so we tested dif ferent biofunctionalized silk fibroin films and com: bination of seeded cells and waye to assemble the 52.4. Creution ofa favoruble environment forthe survival of bolybrid retinas Aatificial 3D retinas structure is achieved by means of the two attached biofilms which allowed contact ctablishment between the neurons and the seeded retinal pigment epithelium cells. The combination. of support cells, molecules and artificial substrates, provided an even more favorable environment for the survival of the neurons and the retinal epithelium cells. We used O1 mm cylindrical molds of witich wwe are first inserting a layer of silk fibroin hydro: gel with mesenchymal cells incaded inside, fllowed ‘witha film with the retinal pigment epithelium cells, a second film with the neurons/Méller cells and finally ‘new layer of silk fibroin hydrogel with mesenchymal cells, We use the hydrogel ax # method to protect and keep the structure together, providing nutrients and creating an environment that isolates theimplant fom the hos that favors the eurvival ofthe implanted cells, As can be sen in figures 6~, celle adhere to the films, However. once the entire sandwich is mounted with the silk fbroin hydrogel, it absorbs a large part of the cells and keeps them inside (figure 8). There fore, the combination of support cells, artificial celle, and substrate provide «favorable environment for the survival of implanted cell, 4. Discussion Retinal degeneration isa leading cause of blindness in the world. Re etiology is complex and involves genetic defects and aging aesociated with stress. In addition to gene therapies for retinal degeneration, cellular therapies have been extensively explored to restore vision in both preclinical animal models and clinical trial, Stem cells from different tissue cources tind theis derived lineages have boon tested to treat et inal degeneration. Currently, itis not known whether visual improvement by cell therapy is due to the fact that the grafted cells are capable of replacing lost retinal neurons; oF duc to the secretion, by these cellalse implants, of neuroprotective and neur trophic fctors that protect the host retina; or both. causes [110]. In replacement approaches for cells lost in the posterior retina, both photoreceptors and retinal pro igenitor cell injected subretinaly manage to migrate tothe correct retinal lamina, form local synapses, and thereforerestore some functionality inanimal models fn, 12OP Paising 7 Neal ig 17 (200) 05008 Nea Dames tal yer srsiyiroge! cP SF-Fim 8 o 1 i ° Not catsimm® 8 Pach Ta rah ang ae a gure ulus of sapporting cll (A) Rpesentv image f MSCs ened on paste (TCP, si lin yop and {Pa ain ls, Se bar 10 pn () tntion of MSCs claro lac and Sb al Soin aogier 2d so 72h in clare (C) Quatieaton af MSCs cate on patie and 6% si bec fi ater 2U and 72 bin ear gute vit cretion ofa 3D mules ti, To provide spe and eesanceo theese scr otic ty is enacd on mi bane are Sp) There erp er amet {Central carn (top) gone lle cre tind conoralwicecopy ge) Oeste este oan tar the {nn are bined (Sp et) they are sre ope the 3D sate tp righ) Alte aac of RPE cle and ‘parent thik hci inn the ral tins cot with the ge + Sc fo et te Bal ler sti (Step op. Right column (Step 4 bot: opti mconcopy mg of cromsecton ofthe msyered bybreins the ‘load the embeded cl wiln thea cin gel ce cen show Recently Liu et al (2020) carried out implant expressing neural, photoreceptor markers, oF syn: experiments of different cell types in the subret-theszing pigment in vivo in the host retina. In inal space of transgenic mice that develop ret- addition, « small part of these cells migrated to inal degeneration. They observed that implanting the layer corresponding to their differentiation, but immortalized sphere stem cells(SDSC)intotheretina the vist majority remained in the injection sites, fave rise to cells that were able to differentiate by So, it appears that, although there is potential fortor asing cy 4 integration, the retinal protection and the improve- rent they observed was mainly due to a parac- rine function of these cells, secreting protective factors, To this parscrine effect, it would be added that the trunsplauted cells do aot survive very lug, and that some tissues (such as the mammalian tal nervous system) have very limited in situ migration [115]. ‘This approach to cell therapy for neurone sch os retinal ganglion cells (RGCs), which require even greater development, axonal growth, and synapto- genesis than other retinal clls (greater complexity of local and distal circuits), is even more complex [114]. Transplanted RGCs have been observed to pro: ject dendrites into the inner plexiform layer (115), and stem cells transplanted directly onto the optic nerve head project a process through it [116]. How ever, transplanted cells located far from the nerve head are unable to extend their axons [117]. The lack of growth and guide factors in adult retinas, added to the reactive envionment present due to the injury or disease suffered, may be the cause of not achieving complete integration and functional- ity of cell transplants, To alter their fate, retinal newr- cons must be encapsulated in a permissive microen- vironment for growth, protect themselves from the diseased or harmful environment, present with cell binding molecules, and be exposed to appropriate ‘mechanical properties to induce and initiate growth [119]. To this way, » finctional integration of the implanted cells could be achieved, In the present wark we show the promising future of silk fbroin as the base of biohybrid retinas. Our principal achievements are (i) fabrication of a ill Sbroin-based layered biohybrid retina (i) primary calture neurons growth and develop neurites on silk fibroin biofilms alone, or (ii) in presence of other cells cultivated on the same biomaterial, () biocom. utility of the biomaterials, (¥) uaatenauce of the Cellular phenotypes and (vi creat jon of a neuropro tective environment through the use ofa silk fibrin. bsed hydrogel. The fact of geting these different lay cece with different cell ypes present inthe retina, giv us the opportunity to create an artificial retina based ‘on cell layers that will later allow the crestion of con. nections between the different cultured cell and thus develop a stratified biobybrid 3D retina with «struc ture similar to the real one (through the pores e would get a ferent ayers). The silk fbroin hydrogel gives consist. fengy support and protection to the ayers with cul tured cells In addition, adding mesenchymal stem cells as suppliers of growth factors to improve the ulture between the cells of the dif viability and increase thecell longevity (Jong,term cel survival) of said cultured cells on the biofilms, ‘We used primary culture cells cellular source closely resembling natural retinas and sllowing us to study the interaction betvreen different cell types ‘with relatively simple experimental protocols. These cells need a suitable substrate for their survival and that facilitates their adhesion. The substrates created are obtained through the functionalization of silk fibroin, which provides binding sites for retinal celle. snd allows sto adapt the substrate for each eell type figure 2. We were able to create the structure ofthe SD retina with two biofilms attached, thus allowing contact between the neurons and the seeded retinal pigment epithelium cellsStctivoin in, G0 Inmunotaing 4.1, How neurons wil integrate with each othert The materials described in thie work not only allow us to create a support, but also to be functional with molecules present inthe extracellular mat fix, becoming the perfect substrate for cell adhes ence and groveth. The presence of laminin in the films will provide us with the creation of « surface where epithelium cells can grow and ean also polae- ized, an essential characteristic fr co-culture and for the epthelivin cells to be able to create connections with other cell types. These types of materials are 10 teh vin lon with PLULAM proves el adatom sd rua Rows 3 ond aaron monly also compatible with the functionslization with other types of molecules such as poly-L-lysine, a necessary component fr seeding and survival of neuronal cell, ‘much more sensitiveto the substrate in which they are seeded, and where itis essential not only the presence ofa good substrate but cavironment fr its survival Previous research has shown that transplant the crestion of favorable ing retinal stem cells into a biodegradable poly ‘mer scaffold increases the rate of cell survival [51]. On the other hand, the dimensional conformationtor asing 1 Nardi 17 (2070) 58008 ‘pectic marker (C; blue) The hydeogel emis forescence with the ste excita or porosity of the scaffold seems to promote the union and subsequent differentiation and orientation of the retinal progenitor cells [119]. Other studies also defend the idea that scaffold topography ingle ences orientation, ait aflects cell adhesion, mor phology, proliferation, differentiation, and migea- Bion [32, 120,121]. Both synthetic polymers and bio- logical materials can be used to generate saffold- ing. Natural materials (such as alginate collagen, te) are suitable to mimic the mative tissue archi tecture [120]. Various synthetic polymers, such as poly--capralactone and poly-d-co-glycalic Llactic acid, offer increneed mechanical strength and control- lable degradation rates [122,123]. However, synthetic polymers tend to cause less biological activity [121] Silks have been extensively studied for tissue engineering applications. The advantages of this materials that silk ibroin is composed, among other amino acids, of glycine, serine and alanine. This com- position alles itt be essly functionalized with spe- cific bioactive molecules to sid cell binding, Once the sericin that coats ibroin is removed, it eaves fibrous material that can dissolve and process in many ways, including transparent films with mechanical proper- ties and degradation behavior that can be tailored to 4 specife applcstion [124] Different studies show how silk fbroin materials can meet the needs of specifi cellular rep airs in the nl cy en engineering of bone, cartilage, adipose tissue, blood vestels and ligaments [125-135], Another application is the generation of artificial nerve guides, as demon: strated (Tang etal 2009). In this stad, the good neuro-compalibility of silk fibroin with hip pocampal neurons, arguing that fbroin is «suitable they observe ‘muteral to treat injuries or diseases of the central nervous system, Sill fbrain has alo been tested in grafts for peripheral nerve regeneration, with results very close to those of autografts [1544 135} “There is not much research using silk fibroin for the growth and implantation of retinal neurone, However, Wittmer et al (2011) [136], eed aligned snd multifunetionaized electrospun elk nanofibers ‘with BDNF and CTNE.Thisscaffolding together with neurotrophic facts significantly increased the sar vival of the RGC and stimulated the growth of its ncurites, which followed the pattern of parallel fibers. “Their results suggest that biomaterials based on bio. logically active (biofinetionslized) silk protein may be good guide support for RGC. However, in thie study, they did not menage to perform implants in retinal explants or in vivo, 0 they did not analyze the migration capacity. Another example is Zhang etal (2015) [4], in which they use seaffolds of silk fibroin and PLCL in a 1:1 ratio. They achieve rapid proliferation of retinal stem cells high cjtocompati lity, and improved diferentiation to specific retinaltor asing MU-PHI (Day 4) (ay 7) (ay 4) (ay 7) (ay 4) (ay 7) neurons in vitro; compared to other scaffolding and control Several studies hveshown that scaffolds from dif- ferent biomaterials allow the development and axonal 118, 121, 136-136) but thi usually occurs in diffrent directions, and extension of RGCe [32, 114, 1 even in a multidirectional way. Tissue engineering, of the nervous system has incorporated soluble and immobilized factors, as well as protein gradients, through a variety of methods {139-142}, promoting, cell survival, axonal cone growth, proper orientation, ete (117) One of the important factors for the success of actifcial constructs is the contribution of an appro- priate cellular environment. Thanks tothe facility of obtaining and high performance, mesenchymal stem 2 IP Merged cells (MSCs) of the bone marrow or adipose tissue, are optimal candidates fora wide variety of applic ations in regenerative medicine sch a8 cell support in the regeneration of the peripheral nervous sys tem, The neurons that make up the retina main. tain their homeostasis thanks to the presence of Miller cells, which participate in the reeyeling of K+, GABA and glutamate ions produced by the intense metabolic activity of the retina. In addition, it has been seen that after damage occurs at the level of the retina, these Maller cells are capable of pro: ducing neurotrophic factore and even proliferate to promote repair. The hydrogel/supporting cells mix ture protects cells and keeps the structure together, provides nutrients and neuroprotective and anti inflammatory molecules while iolating the implanttor asing 1 Neal ig 17202) 055008 from the hostile environment, thus favoring the sus- vival ofthe implanted cells, 4.2, Will the existing dysfunctional Bruch’s ‘membrane impede the normal movement of biological materials and thus impede the function ‘of the implant? ‘An important question is to which extent the existing dysfunctional Bruch's membrane would impede the ‘normal movemnent of biological materials snd thus impede the function ofthe implant. Normally, there ‘would be no impediment to the normal exchange of biological materials nor tothe proper functioning of ‘our biohybrid retins, Our biobybrid aims at repla- ‘ing the damaged retinal tissue. In particula, the silk fibroin biofilm on which we seed pigment epithelial cell will replace the dysfunctional Bruch membrane. ‘Thanks to its thickness and porosity there will be no problems for a correct exchange of biological mater- ials between the host choroid (damaged natura ret~ ina) and the implanted biohybrid one, Pores diamet- cers were in the range of 1-9 jm (Figure 10), very similar to [143]. Pores are small enough to keep the supporting calls (eg, MSCs or Muller eels) into the silk fibroin hydrogel, impeding their diffusion through the host tissue and protecting them from the attcks ofthe immune system. However, they are big enough to allow both, the arrival of metabol- ites to thete cells and the diffusion of MSCx/Miller’s neurotrophic, neuroprotective and neuroregenerst- ive secretome towards the implanted cells and the surrounding host tissue [96, 14]. The sik fibroin hhyeirogel will provide protection against the immune response and structural consistency at the implanta tion time [145]. ‘The shape, adhesion, surface conformation, ‘migestion functions, and wlimately the fate of cells are known to be governed by the properties of cell: bearing substrate. The latter are typically limited to the topography of the substrate surface, its mechan ical rigidity, an the bioactive properties of the sub- strate (ie. theabilty ofthe substrate to target peptides and proteins ina cell) The use of 2D or 3D seaffold- ing has been documented to be an efficient strategy to overcome the limitations of cell transplantation: low cell survival and integration at the traneplant site and difficulties in keeping cells injected into target cals [50] Scaffolds for retinal stem cell (RSC) transplanta~ tion can be freely classified into three different types: ‘rlindrical scaffolds designed to mimic the vertical SD organization of cells in the retina; fibrous scaf- folds designed to mimic the microstructure of the ‘extracellular matrix and hydrogel scaffolds designed to replicate the mechanical properties of the retina Each type has ite advantages and limitations [146]. Cylindrical scaffolds offer orientation of cell growth within the scaffold and cell protection against implantation forces, as well as controlling 1B Nea Dames tal cell differentiation within them, ether by promoting, of inhibiting it. However, the rate of cell migration, from the implant tothe different ayers of the host rt: ins in the few studies in which test are carried out in vivo, isnot very highs having registered a maximum of Jess than 5096 of cells that migrate from the scaffold. In addition, this type of constructions, when made with eynthetic polymers, it ie necessary to function- lize them with adhesion molecules such a laminin Liss} Fibrous scaffolds mimic thestructure ofthe extra cellular matrix. The fibers manage to create a large surface are for cel attachaent while erating intr connected pores for nutrient transport. These strc tures can be chemically and biologically modified ring their construction process; conferring spe cific advantages to these scaffolds. This allows for example, to increase the integration rate of the cells, implanted in the retina, In general, flbrous scaffolds promote better migration of transplanted cells in nat ive and degenerate retinas. However, the cll differen tiation eapacity within this type of scaffolding is less compared to the previous ones [116] In the case of hydrogels, thei stiffness can be adapted to match that of the CNS tissue, In addi tion, there are curtently in situ gelation gel in winch calls are implanted a isolated neurospheres or a5 individual cell suspension; but when they come into contact with the retinal environment, they gel. This property greatly facilitates the implant. However, they havea very limited capacity ta promote the migration. of the transplanted cells to the retina, and the cells ‘mainly migrate to the RPE [46]. Based on the reviewed literature, a fbrain nan. fiber scaffold would allow directional adhesion, pro liferation, and development of retinal neurons, On. the other hand, the characterstis ofthis biomater- {alallow ite functionalization with different biological factors of growth, protection, differentiation, signal ing, ete. This wil allow a saffold to be generated in which celle can differentiate, migrate within i, gen- crate neurites, extend and polarize; obtaining fine tional neurons similar to those found in vivo. On. the other hand, as mentioned above, fibrous eaffolde together with their modifications allow better migra tice of coll to the retina, This characteristic ent be exploited in silk fibers. Ths, together with the fact that silk fibroin allows designs of dtferent degrees of degradability, makes it possible to design a scaffold that will further favor the migration of cll for integ- sation into the retina in vive 43-In vivo injection and the potential problems sand solution that will rise due to this layered hybrid retina ‘The implant of the biohybrid into the eye is not fan easy process due to: () fragility does not allow biohybrids tobe manipulated and introduced into the eye tissue (ii) inflammation provoked to the neuraloP Paising 1 Neal ig 17202) 055008 Nea Dames tal ‘igure 10. Ops micoxopymicrophotoraps showing the fice of he porous bin Sl, Magiicaton OX od TOOK Sue LoS Gab) 38 pn Lissue by the surgical intervention must be controlled and (ii) in order to survive, cellsin the biohybrid ret- ina must be surrounded by a neuroprotective evi ‘onment, For these reasons we decided to weap the Diokybrid up with silk fibroin gel (figure 3) with mes ‘enchymal stem cells and Moller cells encapsulated in, it, two types of cells well known for their neuropro- tective and neuroregenerative secrctome, First of all, the material with which the seat fold is built must comply with the biocompstib ity property to avoid any toxic response or immune reaction [147]. Scalfolds must also be ultrathin (few ‘micrometers thick), implantable, and flexible to void tiesue damage; but mechanically strong to resist Furthermore, in order to promote adequatecelladhe- sion, seaffolds must show sufficient binding signals [148, 149]. On the other hand, these cell transplant ‘constructs must (1) have an enhanced ability to pro- ‘mote cell migration; (2) allow the development of dendrites and axons; (3) lengthening of the Re axons towards the optic nerve head, and (4) regen- erating the axons over long distances in the injured nerve, In addition, possible biochemical and topo- ‘graphic modifications of the material surface must beaded to improve cll differentiation of implanted calls [50] ‘Thanks to the use of silk fbroin, the structures created are completely biocompatible, being entirely inert and not producing an excessive immunclogical ‘ejection. In addition, the different types of formats that can be obtained using silk fibroin-based bioms- terials allows us to use: (i) a substrate easy to manipu- late and to be implanted inthe form of a biofilm, and (Gi) a hydrogel that presents a stifines similar to the ‘one presentin the host tissue, that avoids the rejection ‘of the implant duc to the fexbiity ofthe constrict. ‘The eels we incorporated in the hydrogel sur- rounding the implant enhance the survival of the construct and favor its integration with the host tissue. They serve as a cellular interface between, the implanted cells and the host tissue, and their sscretome protect againet immune nd inflammatory reactions and promote the regeneration of the dam- aged tissues. mn Finally, the preliminary results that we obtained, cellular survival daring the 7 d of our stady and the ‘muntenance of stable configuration in thei layered. structure, suggest that this approach could be vee ful in long-term implantology for AMD treatments Showing the ability to producea stable steuctue that ‘can be subsequently implanted to study its integration, withthe host retina, 4A. Further questions Wmust be acknowledged thatthe implementation of a working silk fiboin- based biohybrid retina entails ‘many addtional challenges beyond the scope of thie study for example, control of cos-inking density and polymer concentration ofthe Bln/el influence the mechanical properties ofthe fl, inchiding pore size and permeability: Mechanical and elastic proper ties should bein therange of native retina and provide stability for cells seed on it in terms of specifica tion (when using undifrenited cells) and fanetion (150). Acuity will depend on the maximum density anal correct organization of neural els that willbe achieved to growth on the biofilm and in particular of photoreceptors, bipolar and retinal ganglion cll Pores diameters, in addition to provide mech- snicalelastic stability, they should allow axons to trowth theough them. Although intuitively this large Pore size should allow the exchanging of soluble Imoleculstfactors responsible of signaling proces, crosslinking density and polymer concentration night differently influence the ability ofthis bioms terial to permeate soluble factors that are required t0 ‘maintain cll survival, axonal guiding and fanctional rewiring. Currently we are working on the temporal dynamics of permeoseectvty of silk fibroin gels to datver molecules in function ofits structure (charge, ‘molecular weight, hydrophobicity). Ii clear that a compromise should be reached Between mechanic allelatic properties for cell fitness and pore size for ‘zonal elongation and delivery of neuroprotectivese- generative fictors that anticipates complexity 'Ad hoc, conteolled silk fibroin degradation another of the important challenges. In the injured central nervous system, peripheral neutrophiloP Paising 1 Neal ig 17202) 055008 and _monocytesimacrophages as well as_resident ‘microglia, are probably responsible for these degrad- stve effects, Degradation depends of inflammatory signals, since a hostile microenvironment exacer Dates the degradation of silk (AMD, brain stroke, etc, manuscript under preparation). At molecular level is largely unknown which proteases might degrade sill froin; although in vito essays identified several proteases (not related t0 the central nervous sys tem) that could degrade silk fibroin in a few weeks. [151]. The identification of precise factor/srespons- iblefor elk fibroin degradation in the central nervous system (and by exteasion in the retina) will allow us to design functionalized sill-based biofilms (for example carrying proteases inhibitors) to control silk fibroin degradation during pre-defined thers peutic time-windovs, It is warthy to mention that silk biodegradation could be useful also for thera peutic purposes, since some silk fbroin byproducts are neuroprotective for neurodegenerative diseases tsa). 5. Conclusion Inthe present paper we provided in vitro evidence for the feasibility of layered biohybrid retinas built with sill silkwour fibroin and cultures of different types ‘of cle retinal epithelium, retinal neural, Miler and mesenchymal stem cells, Silk fibroin-made biofilms have adequate resist- ance, are easly biofunctionalize, act asa perfect up- pport for cell growth and sllow us to crest diferent layers resulting in increasingly complex structures, ‘while maintaining the ability of the seeded cells t0 survive and favoring their possible interaction, Our biokybride are also resistant for physical manipula- tions, that means they are suitable tobe implanted for both, animal experiments and clinical studies. [Next steps are the in vivo testing of the present ‘work and, in parallel, the engineering of silk-based biokybrid retinas with human iPSCs. Also testing 3D bioprinting technologies to speed up the develop ment ofthese retinal prostheses and get closer to the linial spplcstion, Acknowledgments “The authors gratefully acknowledge financial support received from the Spanish Ministerio de Economia, ¥y Competitvidad Spain through grants MAT2016- T68A7-R, MAT2016-79852-R and MAT2015-66666- (C3.3.R and predoctoral FPI grant (AGD) as well as ‘esearch contract (ADAC) from the National Institute for Agricultural and Food Research and Technology (GINIA) and the Spanish State Research Agency (AEI) program INIA-CCAA, from the Comunidad ce Mad tid through grants Neurocentra-B2017/BMD-3760 and IND2018/BMD-9804, FRDF/FEDER Operative 5 Nea Dames tal Program of the Region of Murcia (Project No. 14 20/20). Authors also acknowledge preliminary work (of Rocio Fernandes Sierra (Sill Biomed, CTB-UPM) tnd Rebeca Gallego Ruiz (CTB-UPM, UCM), help in experimental work of Nuria Alfageme Lépes, Nora Serrano Bengoechea, Cristina Castro Domingues, Paula Alvaser Montoya y Miguel Terriza Roberto (UCM, 1418SC) and the invaluable technical support of Soledad Martinez (CTB-UPM).. ORCID iDs Nahla Jemni-Damer @ hps//orcid.org/0000-0002-3518-0262 Daniel Gonzalez-Nieto (@ hutpsd/orcid.org/0000-0003-2972-729% Tose Perez-Rigueiro {@ htps//oreid.org/0000-0001-8298-8308 Michele Papa (© bttps//orcid.org/0000-0002-6609-7453 Fivos Panetsos {© htps://oreid.org/0000-0003-0897-411 References {1} Misa Malesia Kral 2015 Age ead cule Aegeneration = allege frie! pathogen ew {2} Abst and Fowler B 2012 Mechanisms of age elie [p}Lacour Milan | Pand Nias MH 2002 Ageelated cla degnestion Das ging 1910-3 {4} pee RD, Meee WF and an] W200 geese tcl degeneration New BM. 358260017 15] Coen N, Fender Sinche L, Capello, Mane, Delavile tax? and Pina £3014 Calla tesponsee flowing ria injre and theres sppoats foe eurodegenertve diseases Pn, Retinal Ee (Boo) C, Baas DC, Boeke Gongs PC M Pad Bergen A 2010 The dynamic ate of rls ‘membrane Pg Retinal By ex 291-10 tr] Hogan MU, alvrado) Ant Woe} E1971 Hey the Huan sh sand Tb Pid, PS Sunder Publhing) p69? {6} Arden D and Chan “CC 203 Agog isnot dss Songun gested colar depneration om sting Pog, et yee 37 6-29 (91 Dune Durnont My sn andere we (Gaeiee}216 Light senate bein ptwy ond ong Phos bap 33 uo] Piezrello LD 1987 The dies ofthe problem of eye Alsese ston the ely Opal 9 1191-5 [ui] Wooler BS Boulton ME Gatch D and Stenberg 1999 Onive damage and ge etl mnclar degeneration Bo Vic 832 {02} Hamu Anderon€ and Wag $2015 RPE ecroptsiin Feepoe to aidtive sea an in AMD Aging Rew (u3} Bey rat 000 The role eda stresin the Pithogencse of gested maculae dogeneation Sue Opkdainl 45 (us) cts Gan Bouton M E2012 Consagunces of ‘ets Ma 3895171 Neal ig 17202) 055008 115) Lin MT an Bol M2006 Mitochondria dnc snd tian tres neurodegenernive iene Narre {161 Rivers. 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