mouse retinal progenitor cell dynamics on electrospun poly (ϵ-caprolactone)

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Mouse Retinal Progenitor CellDynamics on Electrospun Poly (ϵ-Caprolactone)Sophie Cai a , Meghan Elisabeth Smith b , Stephen MichaelRedenti a c , Gary Edmund Wnek d & Michael JosephYoung aa Department of Ophthalmology , Schepens Eye ResearchInstitute, Harvard Medical School , 20 Staniford Street,Boston , MA , 02114 , USAb Department of Chemical Engineering , Case WesternReserve University , 10900 Euclid Avenue, Cleveland ,OH , 44106 , USAc Department of Biological Sciences , City University ofNew York, Lehman College , 250 Bedford Park BoulevardWest, Bronx , NY , 10468 , USAd Department of Macromolecular Science andEngineering , Case Western Reserve University , 2100Adelbert Road, Cleveland , OH , 44106 , USAPublished online: 11 May 2012.

To cite this article: Sophie Cai , Meghan Elisabeth Smith , Stephen Michael Redenti , GaryEdmund Wnek & Michael Joseph Young (2012) Mouse Retinal Progenitor Cell Dynamicson Electrospun Poly (ϵ-Caprolactone), Journal of Biomaterials Science, Polymer Edition,23:11, 1451-1465

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brill.nl/jbs

Mouse Retinal Progenitor Cell Dynamics on ElectrospunPoly(ε-Caprolactone)

Sophie Cai a, Meghan Elisabeth Smith b,∗, Stephen Michael Redenti a,c,

Gary Edmund Wnek d and Michael Joseph Young a,∗∗

a Department of Ophthalmology, Schepens Eye Research Institute, Harvard Medical School,20 Staniford Street, Boston, MA 02114, USA

b Department of Chemical Engineering, Case Western Reserve University, 10900 Euclid Avenue,Cleveland, OH 44106, USA

c Department of Biological Sciences, City University of New York, Lehman College,250 Bedford Park Boulevard West, Bronx, NY 10468, USA

d Department of Macromolecular Science and Engineering, Case Western Reserve University,2100 Adelbert Road, Cleveland, OH 44106, USA

Received 24 April 2011; accepted 14 June 2011

AbstractAge-related macular degeneration, retinitis pigmentosa and glaucoma are among the many retinal degen-erative diseases where retinal cell death leads to irreversible vision loss and blindness. Working toward acell-replacement-based therapy for such diseases, a number of research groups have recently evaluated thefeasibility of using retinal progenitor cells (RPCs) cultured and transplanted on biodegradable polymer sub-strates to replace damaged retinal tissue. Appropriate polymer substrate design is essential to providing athree-dimensional environment that can facilitate cell adhesion, proliferation and post-transplantation migra-tion into the host environment. In this study, we have designed and fabricated a novel, ultra-thin electrospunpoly(ε-caprolactone) (PCL) scaffold with microscale fiber diameters, appropriate porosity for infiltration byRPCs, and biologically compatible mechanical characteristics. We have verified that our electrospun PCLscaffold supports robust mouse RPC proliferation, adhesion, and differentiation in vitro, as well as migrationinto mouse retinal explants. These promising results make PCL a strong candidate for further developmentas a cell transplantation substrate in retinal regenerative research.© Koninklijke Brill NV, Leiden, 2011

KeywordsProgenitor cell, biocompatibility, electrospinning, scaffold, polycaprolactone, retina

* Present address: NuVention Solutions, Inc., 7650 Hub Parkway, Valley View, OH 44125, USA.** To whom correspondence should be addressed. E-mail: [email protected]

© Koninklijke Brill NV, Leiden, 2011 DOI:10.1163/092050611X584388

Journal of Biomaterials Science 23 (2012) 1451–1465

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1. Introduction

Age-related macular degeneration, glaucoma and retinitis pigmentosa are amongthe many retinal degenerative diseases that cause vision loss and blindness for mil-lions of people worldwide [1–3]. Given that the mammalian retina does not sponta-neously regenerate and current surgical and pharmacological interventions can onlyslow the pathogenesis of retinal degeneration, retinal progenitor cell (RPC) trans-plantation offers a promising therapeutic approach for restoring lost visual function[4, 5]. Multipotent RPCs are a particularly appealing cell source for retinal cellreplacement therapy, because unlike other progenitor or stem cell types, they arealready retinal-lineage-restricted and will not give rise to teratomas upon transplan-tation [6]. Previous research has confirmed that RPCs isolated from developing reti-nae are capable of proliferating in vitro and integrating and differentiating towardmature retinal cell types in vivo upon transplantation [7–9]. However, optimizingRPC delivery and post-transplantation survival remains technically challenging, assubretinal cell injection can often involve significant cell death [8].

Synthetic biocompatible polymer substrates have recently been used to supportRPC adhesion, proliferation and survival before and after transplantation, mark-ing an important advancement in retinal restorative research. Previous work hashighlighted porous three-dimensional polymer scaffolds as superior RPC trans-plantation substrates due to their excellent cell adhesion, localizability, potentialfor promoting differentiation and efficiency of cell delivery to the subretinal space[8, 10, 11]. In an earlier study, the Young lab fabricated a 150-µm-thick poly(L-lactic acid)/poly(lactic-co-glycolic acid) (PLLA/PLGA) biodegradable scaffold anddemonstrated its capacity to adsorb key growth factors and improve RPC post-transplantation survival by 10-fold over single-cell suspension RPC bolus injections[11]. Subsequent work involving other FDA-approved biocompatible polymerssuch as poly(glycolic acid) (PGA), poly(glycerol sebacate) (PGS), poly(methylmethacrylate) (PMMA) and poly(ε-caprolactone) (PCL) has further confirmed theutility of RPC-polymer composites for enhancing cell adhesion, survival and mi-gration after transplantation [12–14].

Among the above-listed polymers, PCL has several properties that render itparticularly well-suited to promoting RPC adhesion and delivery. PCL is a me-chanically compliant material, better withstanding transplantation-induced shearingstresses than more brittle polymers [15]. As a semicrystalline member of the FDA-approved poly(α-hydroxy ester) polymer family, PCL exhibits hydrophobicity anda gradual surface erosion mechanism of biodegradation [8, 12, 13, 16, 17]. Thesecharacteristics improve cell viability and minimize detrimental host environmentacidification caused by the rapid exposure of carboxylic end groups, a problemassociated with polymers like PLGA [8, 12, 13, 16, 17]. In comparison to othercommonly used poly(α-hydroxy ester)s, PCL exhibits remarkable maintenance ofstructural integrity during degradation at physiological conditions, a characteristicimportant for prolonging efficacy as a stable, deliverable substrate [8, 13, 15]. Workby Pritchard et al. and preliminary data from the Young lab have also demonstrated

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that both bulk and electrospun PCL evoke minimal inflammatory response in themammalian retinal environment, demonstrating strong biocompatibility [18, 19].

Building on these favorable properties, in this study we characterized postnatalday 0 (P0) mouse RPC (mRPC) proliferation and integration dynamics on a newlyfabricated ultra-thin, 15-µm-thick electrospun PCL scaffold. We selected the elec-trospinning method of fabrication for the considerable control it offers over polymermorphology, mechanical properties and degradation rates [20, 21]. Electrospinningis particularly useful for generating three-dimensional extracellular-matrix-like ma-trices with variable fiber diameters and high surface-area-to-volume ratios, charac-teristics that support cellular adhesion and proliferation while protecting cells fromtransplantation-induced shearing stresses [20–23].

In customizing the design of our electrospun PCL scaffold, we selected an in-termediate porosity percentage and average pore diameter exceeding the typical10–20 µm range of mRPC diameters, with the goal of promoting nutrient and gasexchange and three-dimensional cellular infiltration without compromising me-chanical strength [8, 22, 24, 25]. We also chose a scaffold thickness of 15 µm tominimize disruption to the 200–300-µm-thick mouse retina while simultaneouslypreserving scaffold rigidity for transplantation purposes [8, 12, 26]. Together, thesecharacteristics promoted robust mRPC proliferation and adhesion without hinderingeventual migration of new cells into host retinal explants, supporting electrospunPCL as a promising scaffold for RPC transplantation studies.

2. Materials and Methods

2.1. PCL Scaffold Fabrication

Poly(ε-caprolactone) (PCL, Mn = 70 000–90 000) was obtained from Sigma-Aldrich. Analytical grade chloroform (CHCl3) was obtained from Sigma Aldrichand used as received.

The electrospinning setup is depicted in Fig. 1a. PCL was dissolved in CHCl3to form a 10 wt% solution. The polymer solution was transferred to a 5-ml syringeattached to a blunt-tipped 18-gauge stainless steel needle, which acted as the sourceelectrode. The syringe was placed in a controllable syringe pump (KDScientific)with a constant flow rate of 1 ml/h applied. A ground electrode, consisting of astainless steel rotating drum, was placed 15 cm from the needle and electrospinningwas carried out through the application of a 15-kV positive voltage to the syringeneedle using a high voltage supply (Spellman). Fibers were collected on the stain-less steel grounded rotating drum until the scaffold reached a thickness of 15 µm,at which point the scaffold was removed.

Scaffold thickness of samples was controlled via the following method: usingconstant experimental setup conditions of solution concentration, voltage, flow rate,separation distance and rotation, mats were produced using given volumes of solu-tion (0.25, 0.5, 0.75 or 1.0 ml). Mat thickness for each solution volume was mea-sured with a digital micrometer at three points to determine the volume of solution

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Figure 1. PCL electrospinning apparatus and product. (a) Schematic of electrospinning setup. Fig-ure not drawn to scale (approximate dimensions: 8-inch-long syringe pump, 4-inch-long syringe,6-inch-long rotating drum, 18-inch-long and 9-inch-high mount). PCL passes from a syringe pumpthrough a highly charged needle for collection as a multilayered three-dimensional matrix on a ro-tating and translating drum. (b) Distribution of 100 electrospun PCL fiber diameters as measuredfrom SEM images. Mean fiber diameter: 2.962 ± 0.894 µm. (c, d) SEM (400× and 1000×) imagesof porous three-dimensional electrospun PCL matrix. (e) Stress–strain profile of electrospun PCL.Young’s (elastic) modulus: 10.513±5.172 MPa; stress-at-break: 2.9105±1.029 MPa; strain-at-break:116.555 ± 39.810% (determined via dynamic mechanical analysis). This figure is published in colourin the online edition of this journal, which can be accessed via http://www.brill.nl/jbs

to be spun to produce a mat of the desired thickness. Samples were then preparedusing this volume. Sample thickness was verified using the digital micrometer, andsamples meeting the target thickness were used in subsequent experiments.

2.2. PCL Scaffold Characterization

Electrospun scaffold morphology was investigated using scanning electron mi-croscopy (SEM; Philips XL30). Samples were sputtered with palladium for 1 minbefore imaging to minimize charging and were investigated at an accelerating volt-age of 5 kV. Fiber diameters were measured from the SEM images of the scaffoldsusing Image J software (NIH). At least 100 random fiber segment diameters weremeasured to generate the average fiber diameter.

Mechanical property analysis of the electrospun mats was performed using dy-namic mechanical analysis (DMA; TA Instruments). Rectangular samples of theelectrospun mats with a 5:1 length:width aspect ratio were loaded into a tensionfilm clamp in the DMA. Using a controlled force, the samples were loaded in ten-sion at 2 MPa/min until failure. All tests were performed at 37◦C, with a preload

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force of 0.01 N applied. Universal Analysis software (TA Instruments) was used todetermine the elastic modulus of the samples, as well as the stress- and strain-at-break.

2.3. Mouse Retinal Progenitor Cell Isolation and Culture

All experiments were performed according to the Schepens Eye Research In-stitute Animal Care and Use Committee and the ARVO Statement for the Useof Animals in Ophthalmic and Vision Research. Isolation of RPCs was per-formed as previously described [11]. Briefly, retinae were isolated from postna-tal day 0 enhanced green fluorescent protein positive (GFP+) transgenic mice(C57BL/6 background). Pooled retinae were dissociated by mincing and digestedwith 0.1% type-1 collagenase (Sigma-Aldrich) for 20 min. The liberated RPCswere passed through a 100-mm-mesh filter, centrifuged at 850 rpm for 3 min,re-suspended in culture medium (Neurobasal (NB); Invitrogen-Gibco) contain-ing 2 mM L-glutamine (Invitrogen-Gibco), 100 mg/ml penicillin–streptomycin(Invitrogen-Gibco), 20 ng/ml epidermal growth factor (EGF; Promega) and neuralsupplements (N2 and B27; Invitrogen-Gibco), and plated into culture wells (Mul-tiwell, Becton Dickinson Labware). Fresh culture medium (2 ml) was added everyother day to the cells and RPC cultures were passaged 1:2 upon reaching 75–85%confluence.

2.4. PCL Scaffold Preparation, FACS, Cell Seeding and Culture

Electrospun PCL scaffolds (2 mm × 2 mm) were incubated in 70% ethanol for24 h and rinsed 3 times with phosphate-buffered saline solution (PBS). PCL scaf-folds were placed into single wells of 24-well culture plates and incubated at 37◦Cin 100 µg/ml mouse laminin (Sigma-Aldrich) in PBS for 1 h to facilitate subse-quent adhesion of RPCs in culture. Scaffolds were then submerged in 1 ml culturemedium and incubated for 1 h at 37◦C. Cultured GFP+ RPCs were dissociatedinto single cell suspensions and seeded onto each laminin-coated PCL scaffold at aconstant seeding density of 10 000 cells per scaffold. RPCs were previously FACS-enriched (Beckman Coulter MoFlo cell sorter, Summit software) for cell viabilityand GFP expression to promote cell population homogeneity. The total volume ofeach well was brought to 2 ml with additional NB media and RPCs were allowedto proliferate on the polymers for 10 days.

2.5. Cell Growth and Proliferation on PCL Scaffold

Expansion of GFP+ mRPCs was analyzed on electrospun PCL scaffolds. To estab-lish a standard mRPC population curve, total mRPC GFP+ signals were detectedin populations (n = 5) from zero to 3.5 × 105 cells in 96-well plates using a Tecan,Genios microplate reader. After establishing a strong linear correlation betweencell count and GFP fluorescence, total GFP emissions from RPCs on 2 mm × 2 mmpolymer squares were recorded daily for 10 days under identical conditions. TheRPC-polymer signals and standard population curve signals were then correlated to


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determine daily cell count on polymer. A Spot ISA-CE camera (Diagnostic Instru-ments) attached to a Nikon Eclipse TE800 microscope was also used to visualizecell proliferation throughout this process.

2.6. SEM of Retinal Progenitor Cells Interacting with PCL Scaffold

Electrospun PCL scaffolds were examined using SEM. Each sample was rinsedtwice in PBS and then soaked in a primary fixative of 3% glutaraldehyde, 0.1 Msodium cacodylate and 0.1 M sucrose for 72 h. The surfaces were subjected totwo 5-min washes with a buffer containing 0.1 M sodium cacodylate and 0.1 Msucrose. The cells were then dehydrated by replacing the buffer with increasingconcentrations of ethanol for 10 min each. The cells were dried by replacing ethanolwith hexamethyldisilazane (HMDS; Polysciences) for 10 min, and subsequentlyair-dried for 30 min. After mounting, the samples were sputter-coated with a 15-nmlayer of gold-palladium at a current of 20 mA and a pressure of 0.05 mbar for 45 s.SEM imaging was conducted on a FEI XL30 Sirion Scanning Electron Microscopeat 5 kV.

2.7. Ex Vivo Retinal Transplantation

C57BL/6 (n = 2) and rhodopsin knockout (Rho–/–) (n = 2) mice were killed andtheir eyes enucleated immediately and placed in ice-cold PBS. The anterior portionof C57BL/6 (n = 4) and rhodopsin knockout (Rho–/–) (n = 4) eyes were removedalong with vitreous. Four radial cuts were made into the posterior eyecup and eachquadrant was flattened, sclera-side down. The flattened eyecup was then cut intofour separate pieces (2 mm × 2 mm) and transferred to a 0.4-µm culture well insert,ganglion side down, and sclera removed. Culture well inserts containing retina wereplaced into 6-well culture plates. Culture medium (500 µl) was added to each culturewell. RPC-seeded PCL scaffolds (2 mm ×2 mm, n = 16 for each mouse type) wereadded to both C57BL/6 and Rho–/– explants and co-cultured for 7 days in culturemedium at 37◦C.

2.8. Immunohistochemical and RPC Migration Analysis of Ex Vivo TransplantedTissue

After incubating retinal explants for 7 days, explants were rinsed 3 times with PBS(warmed to 37◦C), fixed in 4% paraformaldehyde for 1 h, and cryoprotected first in10% sucrose for 12 h and then in 30% sucrose for 12 h. Cryoprotected compositeswere frozen in Optimal Cutting Temperature Compound (Sakura Finetek) at −20◦Cand cut into 40-µm sections using a Minotome Plus (Triangle Biomedical Sciences).For RPC migration analysis, one representative section was taken from each of fiveC57BL/6 and five Rho–/– explants. Sections were imaged using a Nikon EclipseTE800 microscope, after which the number of GFP+ RPCs that had migrated intoeach section was counted. Migration numbers were multiplied by the estimatedtotal number of sections to obtain total migration count, and statistical analysiscomparing the migration counts for C57BL/6 (n = 5) vs. Rho–/– (n = 5) explantswas carried out using the t-test and F -test functions in Excel.


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For immunohistochemical analysis, samples were rinsed 3 × 10 min in PBS andthen blocked and permeabilized in PBS containing 10% goat serum, 1% BSA and0.1% Triton X-100 for 2 h. Samples were incubated in blocking buffer for 12 hat 4◦C with primary antibodies nestin (1:200; BD Biosciences), Crx (1:100; SantaCruz), rhodopsin (1:100; Chemicon), PKCalpha (1:200; Sigma-Aldrich), Recoverin(1:200; Chemicon), Nf-200 (1:400; Sigma), GFAP (1:200; Chemicon) and Ki67(1:200; Sigma-Aldrich). Samples were then rinsed 3×10 min in PBS and incubatedwith a Cy3-labeled secondary antibody 1:100 (Jackson Immunoresearch) for 2 h atroom temperature. Finally, samples were rinsed 3 × 10 min each in PBS and sealedin DAPI-containing mounting medium (Vector Laboratories) for imaging using aLeica TCS SP2 confocal microscope.

3. Results

3.1. Polymer Fabrication, Mechanical Characteristics and Biocompatibility

The electrospun PCL scaffold we fabricated was approx. 15-µm thick with anaverage fiber diameter of 2.962 ± 0.894 µm (Fig. 1a and b). Fibers were ran-domly interwoven, resulting in a porosity of approx. 52%, an average distance of2.84 ± 1.64 µm between pores and an average pore size of 24.4 ± 10.6 µm (largerthan the typical mRPC diameter of 10–20 µm) (Fig. 1c and d). Stress–strain test-ing yielded a Young’s modulus of 10.513 ± 5.172 MPa and elongation-at-breakof 116.555 ± 39.810% (Fig. 1e). While this Young’s modulus is roughly 100-foldhigher than that of native retinal tissue (approx. 0.1 MPa), it is comparable to thatof a previously reported PLGA scaffold; our electrospun PCL scaffold also has anelongation-at-break comparable to that of retinal tissue (83%) [10, 27].

We previously confirmed the biocompatibility of PCL with mammalian retinae(unpublished data). In evaluating the biodegradability of our electrospun PCL scaf-fold, we found that our polymer scaffold lost only 5.2% of its original mass over146 days when constantly stirred in PBS solution at 37◦C. Both these factors favorthe use of PCL in transplantation, as biocompatibility and slow biodegradation pro-long substrate stability while minimizing negative impact on the surrounding hostretinal architecture [24].

3.2. Characterization of mRPC Proliferation Dynamics on Electrospun PCL

Having established the theoretically favorable characteristics of our electrospunPCL scaffold, we next investigated its practical capacity to support mRPC ad-hesion and proliferation. We first established a stable proliferating population ofGFP+ postnatal day 0 (P0) mRPCs in vitro in the absence of polymer. Immunocy-tochemical characterization of these proliferating mRPCs provided evidence for aheterogeneous mRPC population, consistent with previous developmental research[28]. In particular, mRPCs expressed markers both of stemness (Hes1) and reti-nal neuronal fate (Nf-200 and the key photoreceptor markers recoverin and Nrl)(Fig. 2a–d).


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Figure 2. mRPC characterization, FACS viability and GFP expression enrichment, and proliferationon electrospun PCL. (a–d) Isolated mRPCs proliferate as neurospheres in standard culture conditionsand express markers of both stemness: (a) Hes1 and retinal neuronal fate: (b) recoverin, (c) Nf-200,(d) Nrl. Scale bar = 50 µm. (e) In preparation for seeding on electrospun PCL scaffolds, GFP+ mR-PCs were FACS-enriched for viability and GFP expression; a representative FACS image is shownhere. (f) After cell seeding at a density of approx. 10 000 mRPCs per 2 mm×2 mm polymer square onday 0, mRPCs efficiently adhered to the polymer scaffold and proliferated exponentially over 7 days,yielding a final near-saturation density of approx. 177 000 cells per polymer on day 7 (n = 5; SEMerror bars too small to be visible in this graph). Scale bar = 1 mm. This figure is published in colourin the online edition of this journal, which can be accessed via http://www.brill.nl/jbs

After FACS-enriching our mRPCs for GFP expression and viability (Fig. 2e), wemonitored mRPCs grown on laminin-coated 2 mm × 2 mm electrospun polymersquares in nutrient-rich culture media for 10 days. Laminin is an extracellular ma-trix molecule that is regularly used to promote both cell adhesion and neuronal-fateddifferentiation [29]. We first established a strong linear relationship (r2 = 0.9935;data not shown) between cell count and GFP fluorescence intensity, consistent withthe assumption of small variation in mRPC size and GFP expression. We then useddaily GFP fluorescence intensity readouts to track mRPC proliferation on poly-mer over time. Between days 1 and 7 after low-density plating on polymer, themRPCs exhibited an exponential growth pattern, proliferating from an average of10 672 cells on day 1 to 168 130 cells on day 7 (n = 5; growth proportional toe0.4852(Day No.), r2 = 0.9684; Fig. 2f). On day 8, growth slowed significantly, withevidence of single-monolayer saturation as visualized by fluorescence microscopy.The mRPC count reached a plateau on day 9, increasing again on day 10 (data notshown). The irregular growth pattern after day 7 suggests that for single-monolayer


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Figure 3. SEM of mRPC–PCL composites. (a) mRPCs cultured for 7 days on electrospun PCL scaf-fold efficiently proliferate into a monolayer on both sides of the electrospun scaffold. (b) Magnificationof (a) showing mRPC neurospheres adhering to polymer scaffold via neuronal processes. (c) Image ofcentral region of mRPC–PCL composite with relatively low mRPC density; scaffold pores are visible.(d) Magnification of (c) clearly showing cell–cell and cell–polymer contacts during proliferation andpolymer infiltration.

saturation it is sufficient to deliver mRPC-polymer grafts as transplants on day 7of in vitro proliferation (or its equivalent, depending on the initial cell seeding den-sity).

SEM analysis revealed mRPC growth within and around pores throughout theelectrospun PCL scaffold (Fig. 3). mRPCs adhered in monolayers of varying thick-ness to both sides of the polymeric substrate, proliferating as neurospheres whileextending processes across and within the PCL scaffold. This adhesion pattern isparticularly promising, since previous work has suggested that polymeric substratesmay enhance RPC survival and delivery during the transplantation process viaRPC infiltration into the porous matrix, reducing susceptibility to transplantation-induced shearing stresses [8].

3.3. Analysis of mRPC Migration from Polymer Scaffold into Retinal Explants

In addition to testing the capacity of our PCL scaffold to support mRPC prolifera-tion and infiltration, we investigated its effectiveness as a cell delivery vehicle fortransplantation. After 1 week of proliferation, mRPCs adherent to 2 mm × 2 mmelectrospun PCL scaffolds were transferred to the outer nuclear layer of both wild-type (C57BL/6) and rhodopsin knockout (Rho–/–) mouse retinal explants andincubated in culture media for 7 days. Because rhodopsin-containing rods make


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up the majority of the photoreceptors in the mouse retina, we included the Rho–/– mouse type in this study as a useful model for photoreceptor degeneration [13,30]. Previous research suggests that the glial scarring and Muller cell hypertrophypresent in Rho–/– mice can reduce the efficiency of migration of donor cells intothe host retina [13, 30]. In our study, we observed robust migration of mRPCs fromthe polymer into both wild-type (C57BL/6) and Rho–/– retinal tissue, with no sta-tistically significant difference (P = 0.21 by two-tailed unpaired t-test with unequalvariances; unequal variances confirmed by F -test with P = 0.50) between the twoexplant types (n = 5 each; 111 130 ± 21 056 and 95 150 ± 14 722, respectively;Fig. 4a–c). mRPCs integrated in roughly equal proportions into the outer nuclear,inner nuclear, and ganglion cell layers. Although the majority of mRPCs retainedthe round shape characteristic of proliferating progenitor cells, a number of newlyintegrated cells exhibited putative retinal neuronal morphology (Fig. 4d).

Figure 4. Robust migration of mRPCs from electrospun PCL into mouse retinal explants. After 7 daysof RPC–PCL composite culture, RPC–PCL composites were incubated on the outer nuclear layerof wild-type and rhodopsin knockout mouse retinal explants for an additional 7 days. (a) Wild-type(C57BL/6) and (b) Rho–/– retinae both demonstrate robust migration of GFP+ mRPCs from PCL intoeach nuclear layer. PCL, RPC-polymer composite; ONL, outer nuclear layer; INL, inner nuclear layer;GCL, ganglion cell layer. (c) Quantification of number of mRPCs that migrated from polymer intoretinal explants (n = 5 each); no statistically significant difference was observed between the numberof mRPCs that migrated into wild-type and Rho–/– retinae. (d) Magnification from (b) of putativeretinal neuron showing highly developed morphology. Scale bar = 100 µm. This figure is publishedin colour in the online edition of this journal, which can be accessed via http://www.brill.nl/jbs


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Figure 5. Protein expression patterns of mRPCs migrated from electrospun PCL into mouse retinalexplants. Following 7 days of co-culture with mouse retinal explants, mRPCs integrate into retinaltissue and express markers of both pluripotency and maturation. (a) Endogenous Muller cells andmRPCs remaining adherent to PCL polymer express intermediate filament protein GFAP. (b) mRPCsand host retinal neurons express neuronal marker Nf-200. (c) Host photoreceptors and mRPCs lo-calized to the ONL express photoreceptor marker rhodopsin. (d) Host Muller cells and proliferatingmRPCs express intermediate filament marker nestin. Scale bar = 50 µm. Red, primary antibody stain;green, GFP; blue, DAPI nuclear stain. This figure is published in colour in the online edition of thisjournal, which can be accessed via http://www.brill.nl/jbs

Immunohistochemical analysis demonstrated that both mRPCs that migratedinto the retinal explants and those that remained adherent to the polymer expressedboth immature markers (nestin) and mature markers characteristic of retinal neurons(Nf-200), and in particular photoreceptors (rhodopsin) (Fig. 5a–d). Importantly,a number of explanted cells that labeled positively for rhodopsin also localized ap-propriately to the outer nuclear layer, suggesting either that rhodopsin-expressingGFP+ cells were capable of migrating from the polymer to the appropriate retinallocation or that those cells that migrated from the polymer into the outer nuclearlayer received appropriate cues to differentiate into putative photoreceptors. Overalldifferentiation rates appeared low over the 7-day explant period studied, suggestingthat a longer explant period or introduction of additional exogenous factors wouldbe necessary to increase differentiation.

4. Discussion

Given RPCs’ relative ease of in vitro proliferation and demonstrated capacity to sur-vive and integrate with retinal neurons upon transplantation, RPCs are a promising


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source of replacement cells for presently incurable retinal degenerative diseases.Recent work has focused on the development of defined culture media to directdifferentiation of RPCs into particular retinal neuronal types, but there remains thechallenge of developing a transplantation vehicle that can support RPC adhesionand differentiation, be easily localized to areas of retinal tissue damage, and mini-mally disrupt the local retinal environment [31–33]. Our electrospun PCL scaffoldoffers several advantages as an RPC transplantation vehicle.

The 15-µm thickness we selected for our polymer is approx. 3-fold thinner thanthat of most polymers we have previously tested, and it is within the thickness rangethat is thin enough to minimize damage to the subretinal space but also thick enoughto be easily manipulated during transplantation [8, 11, 12, 26]. Our electrospun PCLscaffold also has a porosity percentage within the optimal range for coupling me-chanical strength with biocompatibility — previous research has demonstrated thatoverly porous structures can undermine mechanical integrity, but sufficient porosityis necessary for permitting nutrient and gas exchange, supporting cell adhesion andensuring structural flexibility [14, 15]. Through stress–strain, SEM and proliferationanalysis, we have shown that the mechanical properties and porous 3-D microtopol-ogy of our polymer scaffold support efficient mRPC adhesion and proliferation invitro. It has been suggested that the hydrophobicity of PCL, while beneficial forslowing degradation, can also reduce cell adhesion and proliferation as comparedto more hydrophilic polymers or polymers interwoven with extracellular matrixmolecules [34]. Consistent with the Young lab’s previous work with polymer scaf-folds, in this study we coated our electrospun PCL scaffold with laminin to promoteneuronal differentiation and facilitate adhesion [8, 11, 12]. We conclude from SEMimages and exponential proliferation curves that no serious barriers to cell adhesionand proliferation exist on our laminin-coated electrospun PCL scaffold.

Several factors influenced our decision to employ electrospinning over an alter-nate polymer fabrication method. In an earlier study comparing RPC dynamics onsmooth PCL and nanowire-topology PCL scaffolds, the Young lab demonstratedthe importance of 3-D microtopology in supporting robust cell adhesion [13]. Stud-ies with PCL and other polymers have explored the utility of nanowires, post-likestructures and other specialized surface topologies for promoting cell proliferationand differentiation [8, 12, 13, 35]. While the fabrication procedures involved inthese studies allow very fine precision in polymer fabrication, particularly with re-gard to pore size and spacing, the resulting polymer scaffolds appear too regular toclosely mimic the random topology of retinal infrastructure. We favored the elec-trospinning fabrication method in our study, because in addition to allowing forconsiderable control over polymer design, it produces a more random, multilayeredtopology akin to that native extracellular matrix [36]. Not only does the topology ofour electrospun PCL scaffold more closely mimic that of the native retinal environ-ment, but the multilayered matrix can also allow for the adhesion and proliferationof multiple layers of cells — a feature that has been suggested as an efficient meansof increasing cell delivery without significantly disrupting the host retinal environ-


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ment [35]. Further research is needed to determine the minimum number of cellsneeded for restoring visual function, but if the results of such research indicate thata high cell density of delivery would be ideal, our electrospun PCL scaffold wouldbe well suited for this purpose. For future applications, electrospinning is also par-ticularly valuable for mixing fibers of multiple polymers and/or adapting the PCLscaffold to incorporate natural extracellular matrix components or diffusible adhe-sion, differentiation or trophic factors [21].

From morphological and immunohistochemical analysis of retinal explants cul-tured with mRPC–PCL composites, we observed robust migration of mRPCs fromelectrospun PCL polymer into mouse retinal explants as well as mRPC expressionof various retinal markers after proliferation and differentiation on our polymersubstrate. Notably, we identified expression of photoreceptor-specific markers andappropriate localization of photoreceptor-marker-expressing cells to the outer nu-clear layer. As photoreceptor replacement is an important therapeutic target fordiseases like age-related macular degeneration and retinitis pigmentosa, one impor-tant follow-up question is that of the source of the putative photoreceptors we foundin the outer nuclear layer. If the putative photoreceptors had already expressed pho-toreceptor markers prior to migrating from the polymer to the appropriate targetlocation, this would suggest that future studies should focus on pre-differentiatingRPCs to the appropriate target fate before transplantation. However, if the putativephotoreceptors only expressed photoreceptor markers after migrating to their appro-priate target location, this would suggest that the host retinal environment containssufficient cues to initiate differentiation of appropriately localized RPCs, and futurework should emphasize directed migration of cells from polymer.

The currently accepted model is that the ontogenic state of a cell plays an im-portant role in its capacity to migrate and integrate into the host retinal architecture.MacLaren et al. found that newly postmitotic cells have the highest probabilityof integrating into a host retina upon transplantation and maturing into a location-appropriate retinal cell type [37]. Our lab and several others are, therefore, currentlyworking toward refining the conditions needed to differentiate progenitor or stemcells toward a photoreceptor fate in vitro prior to transplantation. Still, we foundevidence in this and a previous study for nestin immunolabeling in mRPCs that hadmigrated from the polymer into the retina, suggesting that immature cells can inte-grate into the host retinal environment and potentially eventually differentiate intomature retinal cell types [12]. Additional research is, thus, needed to determine therelative importance of predifferentiation and directed migration in influencing finaldifferentiation outcomes.

Going forward, drawing on developmental and directed differentiation studies ofthe various factors contributing to fate-specific retinal differentiation, we anticipatecombining appropriate extrinsic and intrinsic cues with electrospun PCL scaffoldsto facilitate efficient generation of specific retinal cell types [29, 31–33]. Given theslow degradation rate of PCL and its long-term structural integrity, electrospun PCLscaffolds may be especially promising substrates for time-release drug delivery.


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The recent work of Pritchard et al. on the utility of nanoscale electrospun PCLfibers in the porcine retina for facilitating adhesion and transplantation of intactsheets of donor photoreceptors is also encouraging, supporting the considerationof electrospun PCL scaffolds for future in vivo studies toward the clinical goal oftargeted retinal regenerative therapy.

5. Conclusion

We have fabricated a novel ultra-thin electrospun PCL scaffold with appropriatemechanical and structural characteristics for supporting in vitro RPC adhesion,proliferation and differentiation. We have demonstrated that our RPC–PCL com-posite model is capable of delivering high numbers of localized cells to mouseretinal explants, with robust migration, integration, and differentiation. In combina-tion with results from ongoing directed differentiation studies, RPC–PCL-mediatedcell delivery has excellent potential as a practical and flexible therapeutic strategyfor retinal repair.

Acknowledgements

S. C. and M. E. S. contributed equally. We thank James Swift for assistance withSEM imaging and Randy Huang from the Schepens Flow Cytometry Facility forassistance with FACS enrichment. We would also like to thank the National EyeInstitute, Discovery Eye Foundation, and Lincy Foundation for funding support.

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mouse retinal progenitor cell dynamics on electrospun poly (ϵ-caprolactone)

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