Nature-Derived Aloe Vera Gel Blended Silk Fibroin Film Scaffolds for

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Nature-derived aloe vera gel blended silk fibroin film scaffolds for cornea endothelial cell regeneration and transplantation Do Kyung Kim, Bo Ra Sim, and Gilson Khang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b04901 • Publication Date (Web): 31 May 2016 Downloaded from http://pubs.acs.org on June 6, 2016

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ACS Applied Materials & Interfaces

Article

Nature-derived aloe vera gel blended silk fibroin film scaffolds for cornea endothelial cell regeneration and transplantation

Do Kyung Kim, † Bo Ra Sim, † Gilson Khang*, †

† Department of BIN Fusion Technology, Department of Polymer Nano Science & Technology and Polymer BIN Research Center, Chonbuk National University, Deokjin-gu, Jeonju 561-756, Republic of Korea

Corresponding author. Phone: 82-63-270-2848; E-mail: [email protected]

Keywords: aloe vera gel, silk fibroin, scaffold, cornea endothelial cells, tissue engineering

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Abstract: Tissue engineering of cornea support to overcome a shortage of cadaveric corneas for transplantation. The goal of this study was to fabricate an appropriate replacement for cadaveric cornea. In this study, we fabricated transparent ultra thin film scaffolds with naturederived aloe vera (AV) gel and silk fibroin (SF) for corneal endothelial cells (CECs). The scaffolds were subjected to analysis using transparency, contact angle, FESEM, FTIR spectroscopy for its physical and chemical properties. FESEM images revealed that the critical morphology of CECs was formed on the AV gel in the blend with SF than SF alone scaffold. The cell proliferation, phenotype and specific gene marker expressions for CECs were determined by MTT assay, immunofluorescence and reverse transcription polymerase chain (RT-PCR). Incorporation of small amount of AV gel increased the cell viability and maintained its functions well. The scaffolds were easily handled to be transplanted into the rabbit eyes with small incision and examined by its transparency after transplantation and histological staining. The scaffolds attached to the surface of the corneal stroma and integrated with surrounding corneal tissue without significant inflammatory reaction. These results indicate that AV blended SF film scaffolds might be a suitable substitute for alternative corneal graft for transplantation. Introduction Corneal endothelium is the inner single layer of the cornea that is essential for maintenance of cornea thickness, transparency, hydration and its endothelial function derived from neuralcrest that is a barrier between the cornea and anterior aqueous humor.1–3 Corneal endothelium plays the vital role of barrier for metabolic activity and maintains the transparency by using ATPase pump that controls the hydration of stromal.2 The balances are act by the two pumps on each side, one side for transfer nutrients to support keratocytes, and the other opposite side 2


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for transfer the water to prevent the hydration of the corneal stroma.2 Dysfunction of critical pump in cornea results in corneal edema and amblyopia.1 Loss of CECs due to severe injury, surgical intervention, dystrophy or trauma is a critical concern for the human eyes that can lead to blindness.1 Transplantation of clear cornea with penetrating keratoplasty (PK) is essential for clear vision due to limited regenerative ability of human cornea endothelial cells (hCECs) in vivo by interruption of G1-cell cycle and inability of replication of CECs.4,5 Since 1905 when the first corneal transplantation performed in United states, PK is still common surgical procedure. PK, however, is not an ideal therapy for several reasons. PK cannot treat readily for the patients with chemical or thermal burn injuries, other diseases and inflammation.2 Moreover, there are several concerns with keratoplasty that could affect the function of corneal by infection, astigmatism and denervation.6,7 PK requires fresh cadaveric tissue to meet standards and takes weeks or months of the waiting time depending on the patient’s location. PK weaken the structure of the eyes because whole cornea needs incision during the surgery. Patients treated with PK, takes many months to have stabilized vision and heal up.2,6 Thus, this led to significance of efficient alternative corneal graft with high quality materials and tissue engineering strategies. Developing the density and proliferation of corneal endothelium on the artificial cornea with maintaining its function is also crucial.2,8 PK is the most common surgery of keratoplasty worldwide to treat injured cornea with corneal opacification.2 However, over the past several years, new surgical procedure called Descemet’s stripping and endothelial keratoplasty (DSEK) preferably replaced the PK with its more rapid recovery time, faster surgical time, better refractive results and fewer complications after surgery.9 This procedure provides much more stable eye and has fewer 3



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post surgery complications. Patients returns their vision more accurately and rapidly after DSEK treatment than PK.2,10 In the procedure of DSEK, the underlying descemet’s membrane (DM) are physically stripped off with diseased hCECs from the stroma. After removing, the donated cornea tissue including a thin layer of posterior stroma, DM and healthy hCECs is implanted in the patient’s eye.2,9 The isolation and ability of proliferation of progenitor cells on the basal surface of cadaveric cornea is previously reported and cells have been used in animal models. Due to the small incision on cornea, cell loss would be reduced and recovery improved with this technique during the surgical manipulation.11–16 Still, there is a wide range of quality of donor cornea but qualified cadaveric donor tissue with higher density of hCECs is limited worldwide. Selection of a biomaterial for constructing functional artificial cornea that guides CECs with its adhesion, migration and differentiation is primary objective. Scaffolds for corneal endothelium should possess biocompatibility, non-cytotoxicity and biodegradability after in vivo cornea transplantation.2 Especially, artificial cornea should be clear and have aqueous mobility. We chose SF from Bombyx mori (B. mori) for the base material because of easy handling.5 SF offers great biocompatibility, biodegradability, mechanical properties, versatility and transparency in processing for cornea endothelium film scaffolds. SF is natural protein consisting of two main proteins: fibroin and sericin.17 Unlike other natural derived proteins, fibroin, the outer covering protein of SF does not influence immune rejection response. It is reported that fibroin based scaffolds support cell adhesion and proliferation by mimicking the extracellular matrix (ECM).18–20 Studies of diverse cell growth with silk based scaffolds are already described previously and have been used for centuries as a suture. In this study, we used clear gel extracted from AV (Aloe barbadensis Miller) also known as 4


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mucilage, the inner portion of aloe leaves.21 AV is commonly used medical herb for thousand years as it has 75 different medical ingredients like minerals, enzymes, lignin, sugar, salicylic acid and anthraquinones.22 AV gel is popularly known for having various properties, especially for healing burn and wound, enhancing the cell proliferation and differentiation. The whole AV gel extract was the main target for promoting the attachment and proliferation of rabbit cornea endothelial cells (rCECs). AV gained more attention due to its function of anti-inflammatory, anti-fungal, anti-bacterial and anti-arthritic from many decades. AV leaves contain Vitamin C, E and amino acids which inhibit lipid peroxidation and oxidant-induced apoptosis of CECs.21–23 The primary component contained in AV is the polysaccharide acemannan which has positive effect on cellular behavior on dental pulp cells.23,24 Besides biocompatibility and proper transparency for artificial cornea, scaffold materials for cornea regeneration also should possess swelling capacity for cornea hydration.5,25 However, SF typically composed of β-sheet structures because of large hydrophobic domains with short side amino acids chain in the main sequence.17 AV gel contains hydrophilic components which improve water penetration into the SF based films.23 The ultimate goal of this study is to design and implant an efficient transparent alternative corneal grafts with high density of healthy CECs using AV extracted gel and SF. Fabricated SF based films with several concentration of AV gel were analyzed for various properties like transparency, contact angle, FESEM, FTIR, MTT assay, immunofluorescence, RT-PCR, in vivo test etc. Materials and Methods Preparation of SF solution Silkworm cocoons were used to prepare the SF solution. Briefly, silkworm cocoons were cut 5



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and boiled in 0.02 M CaCl2 (Showa Chemical, Japan) with distilled water for 30 min to remove sericin. After boiling, boiled silkworm cocoons were fully dried under the fume hood and dissolved in 9.3 M LiBr (Kanto chemical, Japan) at 60℃ for 4 h. Dissolved solution was R

dialyzed using dialysis tube (Snake Skin○ Dialysis Tubing 3,500 MWCO (molecular weight cut-off), Thermo SCIENCE, USA) for 72 h to remove LiBr. The final concentration of SF solution was 7 wt/vol.%, determined by gravimetric analysis. AV gel extraction Fully grown AV leaves were collected from the garden. Fresh AV leaves were washed with distilled water and rinds were removed. Clear jelly-like pulp was minced in pieces. Fibers were removed by centrifuged at 10000 rpm for 30 min at 4℃. The supernatant was stored at 20℃ and left at the room temperature until use. Fabrication of AV/SF film scaffold SF solution with various proportion of extract AV gel was poured in to a glass dish and was dried fully under the fume hood at room temperature. Dried film scaffolds were treated with methanol for 1 h at room temperature and washed 3 times with distilled water. The thickness of AV/SF film scaffolds was 6-8 um, as evaluated using micrometer (Mitutoyo, Japan). Field Emission Scanning Electron microscopy (FESEM) Cell morphology and attachment of rCECs on the AV/SF films were characterized by field emission scanning electron microscopy (SN-SUPRA 40VP, Carl Zeiss, Germany). Suspension of rCEC was seeded on the film scaffolds (1.9 x104 cells) and cultured for 5 days. The cultured media was changed in every 2 days on 24-well plate. After removing cultured media, scaffolds were washed by phosphate buffered saline (PBS) solution. The adherent 6


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cells were fixed with 2.5% glutaraldehyde (Sigma-Aldrich, USA), and scaffolds were dehydrated using different concentration of ethanol solution (50, 60, 70, 80, 90, and 100%) every 20 min and dried for 24 hr in the room temperature before FESEM analysis. Transparency Transparency of the film scaffolds were measured by spectrum analysis using a SYNERGY R

Mx spectrophotometer (BioTek ○ , USA) at the wavelength range of 380nm - 780nm. Scaffolds without cells and with cells were immersed in PBS before measurement. Fourier Transform Infra Red (FTIR) spectroscopy Infrared spectra of scaffolds were measured using FTIR (Perkin Elmer, USA) in spectra range of 4000 to 400 cm-1 wave numbers. To examine each scaffold for FTIR measurement, samples were prepared without further preparations to be examined directly in the solid and liquid state. Contact angle Hydrophilicity of scaffolds were measured by water contact goniometer (TantecTM, CAMPLUS Micro, USA). Water droplet angle was analyzed between liquid/film interfaces on the AV/SF film. Contact angle was obtained from initial time up to 5 min. Isolation of rCEC and culture New Zealand white rabbits were used to collect the rCECs. Rabbit eyes were extracted and moved to PBS straightly. The surrounding tissues were removed and sterilized with 70% alcohol under the clean bench. Rabbit eyes were washed several times with PBS. Corneal endothelium including DM was peeled off from the rabbit cornea. 0.2% collagenase A 7



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(Roche, Germany) was used to digest the corneal endothelium with DM at 37℃ in a humidified 5% CO2 incubator for 40 min. Digested solution with media was centrifuged at 1500 rpm for 5 min. rCECs were re-suspended in medium containing endothelial growth R

medium-2 (Clonetics○ , USA) with epidermal growth factor, vascular endothelial growth factor, fibroblast growth factor, insulin-like growth factor, hydrocortisone, gentamicin, amphotericin-B, and 10% fetal bovine serum (FBS) and cultured into dishes (Corning, USA). Medium was changed every 2 days. Primary passage 2 of rCECs were used for this study. Initial attachment rCECs (500cells/mm2) were seeded on tissue culture polystyrene (TCP) and SF film with various percentage of AV gel in endothelial basal medium (EBM, Lonza, USA) and cultured for 30 min. After 30 min, cultured media was removed and fixed with cold methanol at 4℃ for 24 h. Samples were rinsed with PBS and stained with DAPI (Santa Cruz Biotechnology, USA). Images were captured by fluorescence microscopy (Nikon Eclipse TE-2000U, Nikon, Japan) and cell nuclear number was counted using Image J program (n=5). Cell proliferation The viability of cultured rCECs was monitored after 4 h, 1 day, 3 days, and 5 days of the culture using MTT(3-[4,-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide;thiazolyl blue) assay. The cultured media was replaced with fresh media before 100 µL MTT solution (5 mg/ml in PBS) was added in to the TCP and the test scaffolds. Samples were stored at 37℃ in a humidified 5% CO2 incubator for 3 h to allow formation of formazan crystal. Later the supernatant was removed and 1 ml of dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals. The dissolved solution was placed to read in 96-well plate, and absorbance 8


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has been recorded at 570nm. mRNA expression Total ribonucleic acid (RNA) was extracted from CECs cultured for 5 days on TCP and AV/SF scaffolds. Cultured CECs were washed with PBS and treated with TRIzol reagent (Takara, Japan) according to the manufacturer’s instruction. Extracted RNA samples were quantified using Eppendorf BioSpectrometer (Eppendorf, Germany). The expression of mRNAs of TCP and SF film with various concentration of AV gel were confirmed by related genes such as Aquaporin-1 (Aq-1), Na+/K+-ATPase (NaK), Chloride channel protein 3 (CLCN3), Volt-age-dependent anion channel 2 (VDAC2), Volt-age-dependent anion channel 3 (VDAC3) and Collagen type VIII (COL 8). Genes were evaluated by RT-PCR. Samples were denatured for 30 s at 95℃ and 1min/kb elongation at 72℃. Products of polymerization chain reaction were separated by electrophoresis at 100 V on 0.7% agarose gel (Lonza, USA) in 0.5% TAE buffer (Showa Chemical, Japan) and visualized using ethidium bromide (Sigma-Aldrich, USA). Histological Analysis The identity of rCECs was shown by the expression of NaK. The histological expression of rCECs was monitored after 5 days of culture. rCECs were fixed with 4% of formaldehyde at 4℃ and placed for 24 h at room temperature. After discard the fixed solution and wash with PBS for 3 times, protein blocking solution (DAKO, Denmark) was added for 12 min under dark room at room temperature. Fixed samples were incubated with anti-NaK (1:300, Santa Crux Biotechnology, USA) for primary antibody overnight at 4℃. As secondary antibodies, Fluorescein-labeled goat anti-mouse IgG (1:300, Santa Cruz Biotechnology, USA) were used 9



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for NaK detection. At last, mounting medium with DAPI (Santa Cruz Biotechnology, USA) was used for mounting and the immunofluorescence images were taken by confocal laser scanning microscope (LSM 510 META, Carl Zeiss, Germany). In vivo test – Transplantation of cultivated rCECs on AV/SF films into rabbit eyes Nine female New Zealand White rabbits weighing 400-500 g were used in the experiment. Rabbits were anesthetized intramuscularly 5 cc for each rabbit with a mixture of Alfaxalone (4 mg/kg, Jurox, Australia) and Domitor (1 mg/kg, Orion, Finland). The 5 mm of cornea limbal area was penetrated with slit knife and air was injected into the anterior chamber. The bent 20 G needle was used to scrape DM in circular shape with diameter of 7 mm. Cornea endothelium and DM were removed by mechanical scraping using a reverse sinskey hook. To prepare the scaffolds for implantation, scaffolds with a diameter of 6 mm were cut with biopsy punch (Kai industry, Japan). Isolated rCECs were cultured on the surface of the scaffolds to be confluent. The rabbits were divided into three groups. Frist group (control, 3 rabbits) was injured by scrape off DM without receiving any treatment. Second group (transplant, 3 rabbits) was injured and received implantation of 0 wt.% AV/SF film scaffold with healthy rCECs. Third group (transplant, 3 rabbits) was injured and received implantation of 3 wt.% AV/SF film scaffold which was the most superior group among the experimental groups in vitro. After surgery, one dose of Oxytetracycline dihydrate (2 mg/kg, Eagle Vet, South Korea) was given intramuscularly. Levofloxacin 0.5% (Samil, South Korea) drops were applied daily when sign of inflammation was observed. Cornea photographs were taken of each rabbit at the time point. All rabbits were sacrificed at 4 weeks and whole cornea was harvested and fixed in 10 % formalin solution (Sigma-Aldrich, USA) for prepare histological and immunohistological analysis. 10


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Biocompatibility of the scaffolds in the rabbit anterior chamber After 4 weeks of post surgery, corneal photographs were taken of each animal and the rabbits were sacrificed. Sacrificed rabbit eyes were harvested and fixed with 10 % formalin solution. Samples were embedded in paraffin to make blocks. The paraffin blocks were sectioned into 7㎛ and hematoxylin and eosin (H&E) staining was carried out.

For immunohistochemistry analysis, paraffin sections were deparaffinized and stained with NaK and zona ocludin-1 (ZO-1), by standard immunohistochemistry. Protein blocking solution (DAKO) was added for 12 min under dark room at room temperature. After nonspecific blocking, samples were incubated with anti-NaK (1:150, Santa Crux Biotechnology, USA) and ZO-1 (1:100, Santa Crux Biotechnology, USA) as primary antibodies for 90 min at R

37℃. As secondary antibodies, Alexa Fluor○ 594-conjugated AffiniPure Donkey Anti-Rabbit IgG (1:300, Jackson Immuno Research Laboratories, Inc., USA) and Fluorescein-labeled Goat Anti-mouse IgG (1:300, Santa Cruz Biotechnology, USA) were used for NaK and ZO-1 detection. Lastly, samples were mounted with mounting medium with DAPI (Santa Cruz Biotechnology, USA) and cornea endothelium immunofluorescence images were taken by confocal laser scanning microscope (LSM 510 META, Carl Zeiss, Germany). Statistical analysis All results are presented as mean±standard deviation (SD). Statistical analysis was carried out based on Student’s t-test (Excel 2016, Microsoft) and the differences were considered significant at P

Nature-Derived Aloe Vera Gel Blended Silk Fibroin Film Scaffolds for

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