May 31, 2016 - derived aloe vera (AV) gel and silk fibroin (SF) for corneal endothelial cells (CECs). The scaffolds were subjected to analysis of transparency ...
May 31, 2016 - Do Kyung Kim, Bo Ra Sim, and Gilson Khang*. Department of BIN Fusion Technology, Department of Polymer Nano Science & Technology, ...
May 31, 2016 - Nature-Derived Aloe Vera Gel Blended Silk Fibroin Film Scaffolds for Cornea Endothelial Cell Regeneration and Transplantation. Do Kyung Kim, Bo Ra Sim, and ... Interfaces , 2016, 8 (24), pp 15160â15168 ... Citation data is made avail
May 31, 2016 - 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. ACS Appl. Mater. Interfaces , 2016, 8 (2
May 31, 2016 - Cornea Endothelial Cell Regeneration and Transplantation. Do Kyung Kim ... The corneal endothelium is the inner single layer of the cornea.
May 31, 2016 - Nature-Derived Aloe Vera Gel Blended Silk Fibroin Film Scaffolds for ... Emily A. Gosselin , Tess Torregrosa , Chiara E. Ghezzi , Alexandra C.
Jul 26, 2016 - Real-time PCR analysis further confirmed up-regulation of cartilage-specific aggrecan, sox-9 (â¼1.5-fold) and collagen type II (â¼2-fold) marker genes (p â¤ 0.01) in blended hydrogels. The hydrogels demonstrated immunocompatibility,
Jul 26, 2016 - Real-time PCR analysis further confirmed up-regulation of cartilage-specific aggrecan, sox-9 (â¼1.5-fold) and collagen type II (â¼2-fold) marker genes (p â¤ 0.01) in blended hydrogels. The hydrogels demonstrated immunocompatibility,
Jul 26, 2016 - ABSTRACT: An osteoarthritis pandemic has accelerated exploration of various biomaterials for cartilage reconstruction with a special emphasis ...
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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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
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
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
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
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
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
(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
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
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
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<0.05(*), P<0.01(**), P<0.001(***). Result and discussion
Transparency Transparency of fabricated SF scaffolds blended with several concentration of AV gel was analyzed by spectrophotometer at the wavelength range from 380nm to 780nm. Transparency is crucial factor of cornea substitute not only for recovery of patient’s vision but also for monitor the behavior of the cells, infection and process of healing.5,26 Transparent artificial cornea with cultured healthy CECs will provide efficient transplantation of corneal endothelium. The graph (Fig. 1) shows the transparency of the SF films blended with several concentration of AV gel on with/without cell culture. TCP (commercially available material) as a positive control, shows high transparency on with/without cells. Transparency of 0 wt.% AV/SF and 1 wt.% AV/SF film scaffolds had not significant difference. As well, 3 wt.% and 5 wt.% AV/SF also show similar transparency and have higher transparency than of 0 wt.% and 1 wt.% AV/SF scaffolds. After 5 days cell culture, the transparency of AV/SF scaffolds were in the optical intensity order of TCP<0 wt.% AV/SF<1 wt.% AV/SF<3 wt.% AV/SF<5 wt.% AV/SF. As more clear AV gel was incorporated in SF solution, the scaffolds showed higher transparency. This is may due to the component in the scaffold of high-purify inner part of AV leaf and its purity effect.27 Transparent AV/SF scaffolds not only provide positive effect on clear vision for patients but also for monitoring the behavior of CECs during the repair time after implantation. The optical intensity of human acellular cornea stroma had between 0.1 and 0.13 at the wavelength range of 400nm and650 nm and over 0.1 only at the higher wavelengths than 700 nm “far red”. Compare to the optical intensity of acellular human cornea stroma, optical intensity of AV/SF film scaffolds had between 0.06 and 0.17 at the wavelength range of 400 nm to 650 nm without cell seeding.2 Thus, the transparency of AV/SF film scaffolds has 12
accepted value for the cornea substitute for transplantation. For further confirmation, inset of Fig.1 shows the image of transparent AV/SF scaffolds.
Fig. 1 Transparency results of the TCP, different concentration of AV/SF scaffolds in the wavelength of 380nm to 780nm without cell (A) with confluent cell (B). Inset image shows the optical images of the scaffolds. Hydrophilicity of the films Hydrophilicity of each scaffold was evaluated at an initial time (0 min) to final time (5 min). Hydrophilicity/hydrophobicity of the surface influences the cell migration, proliferation and adhesion on the scaffolds.5 The contact angle of micro droplet on each scaffold for cornea regeneration was measured to confirm the possibility of AV blended with SF. Contact angle account has been reduced to increase water absorption of material. The contact angle result of AV blended scaffolds show better hydrophilicity as more AV added than the control scaffold in the order of 0 wt.% AV/SF<1 wt.% AV/SF<3 wt.% AV/SF<5 wt.% AV/SF. (Fig. 2(A)) Hydrophilic film scaffolds maintain moist environment, preventing bacterial infections and stimulating cell migration.28 Moreover, hydrophilic properties of scaffold is related to the
prevention of losing body fluids and essential nutrients in vivo.29 Thus, this scaffolds can be suggested as a suitable graft for tissue specificity and cellular interaction. FTIR FTIR spectroscopy used for analyzing the crystallization of SF and to defining the composition of each scaffold. The graph (Fig. 2(B)) shows crystallized SF at the range of 1482-1705cm-1. β-sheet formation is shown by three amid peaks at 1644 cm-1(C=O stretch) amide-I, 1516 cm-1(N-H bend) amide-II and 1270 cm-1 (C=N stretch) amide-III, respectively.30 Absorption band of AV due to hydroxyl group in the amino acids which is O-H stretch by hydrogen bond had strong intensity at 3341 cm-1. The absorption band at 1644 cm-1 is the carboxyl group (asymmetrical COO− stretching vibration or O–H deformation vibration of H2O ) in polysaccharides of AV gel.
The characteristic peaks of SF are
observed to have shallower depth with content increasing of AV gel. The reduction of intensity is may due to formation of hydrogen bond between AV and SF and confirming the successful incorporation between AV gel and SF to the polymeric base.32 It has been reported inter-hydrogen bonds between two different macromolecules are stronger than the same polymer molecules.33 The broad peaks between 3000 and 3500cm-1 respond to –OH stretching and physical adsorption moisture of film surfaces.
Fig. 2 (A) Contact angle of single droplet on TCP, different concentration of AV/SF scaffolds up to 5 min (n = 5). (B) FTIR results of AV/SF scaffolds and AV gel. Initial attachment Initial attachment of the rCECs is important for corneal regeneration as it affects the proliferation and differentiation of the corneal endothelium. The substrate that provides the better condition for initial attachment means its affinity towards the cells on the specific substrates.34–36 High rate of initial attachment defines not only the proliferation but also it reduces the time for cell to reach its confluence. Reducing the time span will influence the donor cornea sample preparation and to adapt after surgery ideal cell density. As the number of initial attachment decreases it lose the function of the corneal endothelium.5 Therefore, protecting the cornea from the outside negative factors is important because of limited proliferation ability of corneal endothelium in vivo. Fig. 3(A) shows initial attachment of rCECs after seeding with density of 2000 cells/mm2 for 30 min. The initial attachment on 3 wt.% AV/SF (344 ± 28 cells/mm2) shows slightly higher initial attachment than on 0 wt.% AV/SF (326 ± 22 cells/mm2) and 1 wt.% AV/SF (327 ± 42 cells/mm2) but was not apparent. Compared to TCP, initial attachment on 0, 1 and 3 wt.% AV/SF scaffold show higher initial 15
cell attachment than 5 wt.% (236 ± 13 cells/mm2 ) AV/SF scaffold. This result implies AV/SF composite scaffold offers desired initial adhesive property for corneal endothelium. Cell proliferation Proliferation of CECs on the scaffolds for transplantation is crucial for vision recovery. When the density of CEC reaches the minimum functional level around 500 cells/mm2, CEC loss its function and edema start to occur.4 Loss of CECs or damages only repaired by the spreading of the existing CECs.2,5 Moreover, CECs cannot divide or repair itself during adulthood.2 As a positive control, TCP shows a high cell proliferation. The proliferation of rCECs in different proportion (1, 3, 5 wt. %) of AV gel with SF scaffolds were evaluated by MTT assay seeding after 4h, 1, 3, 5 days after. (Fig. 3(B)) There is no significant difference on cell proliferation between the experimental groups up to 1 day of culture. After 1 day, 3 wt.% AV/SF scaffold showed remarkable increase compare to experimental groups. rCECs on SF scaffold with 1 wt.% and 3 wt.% extracted AV gel show higher cell growth than the scaffold without AV gel blended. At the 5 days of culture, cell proliferation on 3 wt.% AV/SF shows the highest rate compare to the proliferation rate on the other scaffolds. The main components of AV such as glycoprotein, emodin and aloesin has been reported to promote the cell proliferation activity.23 However, proliferation on 5 wt.% AV/SF scaffold was significantly lower than the control and other experimental groups after 3 days of culture. The proliferation of CECs on 3 wt.% AV/SF scaffold was two fold higher than proliferation on 5 wt.% AV/SF scaffold at the day 5. Same has been well explained by the hypersensitivity reactions of AV on the cell proliferation.37,38 Proliferation results suggest the incorporation of proper amount of AV into SF film scaffolds could support the cell proliferation.
Fig. 3 (A) Initial attachment of rCECs after 30 min seeding in serum free medium (n = 3). (B) rCEC proliferation result by MTT assay in EGM-2 (n = 3), P<0.05(*), P<0.01(**) Surface properties of films and morphology of rCEC on the film Morphology characteristic of corneal endothelium substrate is important for flexibility, regeneration of corneal endothelium and cell interaction.8 FESEM analysis (Fig. 4) shows the surface roughness of the films without cell seeding. The pristine SF film however, the smoothness is not much significant. The scaffolds show little more rough surfaces as concentration of AV gel in SF was increased. This may due to the hydrophilic components of AV and SF have intermolecular interaction between two materials. Morphology of rCECs on each scaffold was evaluated by FESEM images after cultured for 5 days. Compared to the other experimental groups, FESEM image of the 3 wt.% AV/SF scaffold show an appropriate polygonal like morphology with ECM interaction in vitro that regulates hydration of stroma and anterior chamber that maintains the cornea transparency in vivo.26 The cell binding components of ECM supports the formation and maintenance of the monolayer.39
Fig. 4 FESEM images of the surfaces of the scaffolds and rCEC morphology on the TCP and scaffolds. mRNA Expression To confirm the expression of mRNAs, diverse gene markers for rCECs such as Aq-1, NaK, CLCN3, VDAC2, VDAC3 and COL 8 were used. (Fig. 5) Aq-1 is the important marker for fluid transportation that increases penetrability of the water molecule.40 NaK facilitates for maintain the transparency through controls the edema of cell substrate and pump function.41 CLCN3 plays the role of pH regulator, cell-to-cell immigration, differentiation and proliferation and transporting organic molecules. CLCN3 is especially important in keeping proper shape and size of rCEC. VDAC2 and VDAC3 regulate interactions between the small molecules and protein among the cells.42 COL8 regulates the amount of fluid in cornea for clear vision.43 Overall, all gene markers were well expressed and normalized with by β-actin. Compared with SF scaffold, most of the genes analyzed higher expression rate in the group of 3 wt.% AV/SF scaffold. rCECs on the 3 wt.% AV/SF shows highly enhanced expression of Aq-1, CLCN3, VDAC2 and VDAC3. Compared to TCP, fabricated SF based scaffolds are favorable environment for typical gene expression of CEC, especially, 3 wt.% AV/SF 18
provides for a prominent role in cellular interaction, cell adhesion and proliferation. Hence, AV/SF scaffolds provide useful avenues for clinical carriers with maintaining the phenotype of CECs.
Fig. 5 Specific gene expressions of rCEC by RT-PCR. (normalized by β-actin) Histological Expression The expression of NaK, the typical CEC marker, indicates importance of sodium potassium pump for purifying water from corneal stroma. Transportation of water from corneal stroma to CEC and nutrient balance are essential for clear vision.41,44,45 There are significant differences on cell numbers and morphology of rCECs on each substratum at 5 days after seeding. (Fig. 6) Morphology of rCECs are observed to have directions with fibrous morphology and unreached confluence except for 3 wt.% AV/SF scaffold. On the 3 wt.% AV/SF, rCECs are tightly connected with characteristic polygonal shape of rCECs and have tight cell junctions that control its hydration by pump through pump leak system. Figure. 3 demonstrates 3 wt.% AV/SF scaffold support CECs to grow better and does not impair the 19
CEC binding characteristics. The scaffolds with proper amount of AV gel incorporated with SF can be envisioned as promising rCEC carrier for transplantable intraocular cell delivery.
Fig. 6 Immunofluorescence microscopy images of rCECs with staining of NaK. In situ implantation of scaffolds- Histological and Immunohistochemistry analysis In vivo test responses to AV/SF scaffolds were evaluated by performing implantation in the anterior chamber of the rabbits’ eyes. Although, we focused on the in vitro test of the scaffolds such as ability of rCEC’s attachment, proliferation and remaining its functions well. One of our goal of the studies was to fabricate obtainable scaffold with minimal inflammatory response and easily transplantable CEC carrier for transplantation. CECs have been seeded on variety materials including human amniotic membrane1,46, collagen sheets47– 51
, DM51,52. However, many scaffolds had trouble with inserting during the transplantation
surgery due to its mechanical properties and difficulty with handling. Animal experimentation is important to test biocompatibility aspects of the scaffolds and avoid acute and chronic rejection response after implantation. All 6 rabbits received implantation had clearing cornea than of control. There was difference between the group implanted SF film scaffold with and without AV gel blended. After 4 weeks of implantation, the group received 3 wt.% AV/SF scaffold with rCECs revealed higher corneal transparency compared to SF film with rCECs. (Fig. 7(B)) The transplanted eyes were processed for histological examination by H&E 20
staining. (Fig. 7(C)) H&E staining results show DM was fully removed and the scaffolds were well adhered to the rabbit cornea stroma. Moreover, some integration between the scaffolds and the surrounding tissues were observed without inflammatory responses. Immunohistology with NaK and ZO-1 staining of the rabbit cornea and scaffolds demonstrate that scaffolds and rCECs were attached well with its function. (Fig. 8(A))
carried out NaK and ZO-1 staining on the stripped off DM carrying rCECs to confirm the surgical procedure was well proceeded. (Fig. 8(B)) All rabbits were lack of inflammatory response surrounding intraocular through the course of experiment. In vivo results demonstrate that bioengineered AV/SF film scaffolds have capacity of self CEC regeneration and biocompatibility after transplantation.
Fig. 7 (A) Schematic image of the DSEK surgical procedure. (B) Transplantation of SF based film scaffolds. The DM with rCECs were mechanically removed and scaffolds were implanted. Image of un-transplanted (Control), transplanted scaffold of 0 wt.% AV/SF (SF) and 3 wt.% AV/SF (AV/SF) film scaffolds with rCECs after 4 weeks of post surgery. (C) 21
H&E staining of transplanted eyes show adherent scaffolds on the surface of the corneal stroma with attached rCECs. (scale bar = 50 ㎛)
Fig. 8 (A) Immunofluorescence images of un-transplant (Control) and transplanted 0 wt.% AV/SF (SF) and 3 wt.% AV/SF (AV/SF) film scaffolds in the rabbit corneal anterior chamber after 4 weeks of post surgery with staining of NaK and ZO-1 (scale bar = 100 um). (B) Immunofluorescence images of stripped DM with rCECs.
Conclusion Transparent AV/SF ultra thin film scaffolds were well fabricated and functionalized to support rCEC proliferation to be transplanted. The AV/SF scaffolds described here are made with nature-derived biomaterials SF and AV gel by physical (drying) and chemical (crosslinking) processes. We fabricated clear and ultra thin film scaffolds with the desired properties described above. It has been demonstrated that thin grafts are more suitable for blurred visual recovery with rapid heal up time and minimal corneal refraction alteration.5,53 The fabricated scaffolds were highly transparent with its biocompatibility to be transplanted into the anterior chamber of the eye. Fabricated scaffolds provide favorable environment to rCECs with maintaining their morphology and critical functions. Moreover, implantation of scaffolds into the rabbit eyes was successfully done and scaffolds were well attached to corneal stroma without any significant inflammatory responses. Results demonstrate composite AV/SF scaffolds compared to the pristine SF scaffolds may provide efficient carrier for patients with various corneal diseases. Acknowledgements This research is supported by Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Korean government (MEST) (NRF2012M3A9C6050204).
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