Bruch's-Mimetic Nanofibrous Membranes Functionalized with the Integrin-Binding Peptides as a Promising Approach for Human Retinal Pigment Epithelium Cell Transplantation.

BACKGROUND: This study aimed to develop an ultrathin nanofibrous membrane able to, firstly, mimic the natural fibrous architecture of human Bruch's membrane (BM) and, secondly, promote survival of retinal pigment epithelial (RPE) cells after surface functionalization of fibrous membranes. METHODS: Integrin-binding peptides (IBPs) that specifically interact with appropriate adhesion receptors on RPEs were immobilized on Bruch's-mimetic membranes to promote coverage of RPEs. Surface morphologies, Fourier-transform infrared spectroscopy spectra, contact angle analysis, Alamar Blue assay, live/dead assay, immunofluorescence staining, and scanning electron microscopy were used to evaluate the outcome. RESULTS: Results showed that coated membranes maintained the original morphology of nanofibers. After coating with IBPs, the water contact angle of the membrane surfaces varied from 92.38 ± 0.67 degrees to 20.16 ± 0.81 degrees. RPE cells seeded on IBP-coated membranes showed the highest viability at all time points (Day 1, p < 0.05; Day 3, p < 0.01; Days 7 and 14, p < 0.001). The proliferation rate of RPE cells on uncoated poly(ε-caprolactone) (PCL) membranes was significantly lower than that of IBP-coated membranes (p < 0.001). SEM images showed a well-organized hexa/polygonal monolayer of RPE cells on IBP-coated membranes. RPE cells proliferated rapidly, contacted, and became confluent. RPE cells formed a tight adhesion with nanofibers under high-magnification SEM. Our findings confirmed that the IBP-coated PCL membrane improved the attachment, proliferation, and viability of RPE cells. In addition, in this study, we used serum-free culture for RPE cells and short IBPs without immunogenicity to prevent graft rejection and immunogenicity during transplantation. CONCLUSIONS: These results indicated that the biomimic BM-IBP-RPE nanofibrous graft might be a new, practicable approach to increase the success rate of RPE cell transplantation.

A novel approach for subretinal implantation of ultrathin substrates containing stem cell-derived retinal pigment epithelium monolayer.

OBJECTIVE: To evaluate the feasibility of a new technique for the implantation of ultrathin substrates containing stem cell-derived retinal pigment epithelium (RPE) cells into the subretinal space of retina-degenerate Royal College of Surgeon (RCS) rats. METHODS: A platform device was used for the implantation of 4-µm-thick parylene substrates containing a monolayer of human embryonic stem cell-derived RPE (hESC-RPE). Normal Copenhagen rats (n = 6) and RCS rats (n = 5) were used for the study. Spectral-domain optical coherence tomography (SD-OCT) scanning and histological examinations were performed to confirm placement location of the implant. hESC-RPE cells attached to the substrate before and after implantation were evaluated using standard cell counting techniques. RESULTS: SD-OCT scanning and histological examination revealed that the substrates were precisely placed in the rat's subretinal space. The hESC-RPE cell monolayer that covered the surface of the substrate was found to be intact after implantation. Cell counting data showed that less than 2% of cells were lost from the substrate due to the implantation procedure (preimplantation count 2,792 ± 74.09 cells versus postimplantation count 2,741 ± 62.08 cells). Detailed microscopic examination suggested that the cell loss occurred mostly along the edges of the implant. CONCLUSION: With the help of this platform device, it is possible to implant ultrathin substrates containing an RPE monolayer into the rat's subretinal space. This technique can be a useful approach for stem cell-based tissue bioengineering techniques in retinal transplantation research.

Case-control study of risk factors for no light perception after open-globe injury: eye injury vitrectomy study.

PURPOSE: Investigate possible risk factors of no light perception (NLP) after open-globe injury. Explore whether these risk factors are predictors for an unfavorable visual outcome. METHODS: This case-control study matched 72 eyes with NLP according to type and zone of injury to 2 controls per case with light perception or better vision. Cases were selected from the Eye Injury Vitrectomy Study database. All injured eyes in the study underwent surgical intervention. RESULTS: Ciliary body damage (odds ratio = 2.94), closed funnel retinal detachment (odds ratio = 2.43), and choroidal damage (odds ratio = 2.80) were independent risk factors for NLP after open-globe injury. There were 67 traumatized eyes with NLP that had ≥1 of these risk factors. In 43 of the cases (64.2%), the eyes recovered light perception or better after vitreoretinal surgery. The five traumatized NLP cases without these risk factors obtained a favorable visual outcome after vitreoretinal surgery. There was no statistical significance in visual outcome between them (P = 0.162). CONCLUSION: Ciliary body damage, closed funnel retinal detachment, and choroidal damage are independent risk factors for NLP posttrauma but not prognostic indicators for NLP visual outcome. Traumatized eyes with NLP may recover light perception or better vision if appropriate interventional measures are used for treatment of the injured ciliary body, retina, and choroid.

In vitro and in vivo evaluation of a small-caliber coaxial electrospun vascular graft loaded with heparin and VEGF.

INTRODUCTION: To date, clinically available expanded polytetrafluoro-ethylene (ePTFE) vascular grafts are suboptimal for reconstructing small caliber (D < 6 mm) arteries, owing to thrombosis in early and restenosis in late stage. Our aim in this preliminary study was to fabricate a nano-fibrous vascular graft which was biofunctionalized with VEGF165 and heparin. The short term performance was evaluated both in vitro and in vivo. METHOD: Four-mm caliber grafts were prepared by the coaxial-elctrospun technique, which consisted of poly(l-lactide-co-caprolactone) [P(LLA-CL)] collagen and elastin. Heparin and endothelial cell growth factor-165 (VEGF165) were encapsulated in the core of the fibrous. Controlled release of the heparin and VEGF165 were evaluated for 28 days. Endothelial cells were cultured on the electrospun grafts or ePTFE grafts as controls. The cellular adhesion, proliferation and morphology were examined. Electrospun or ePTFE grafts were randomly implanted into a rabbit infrarenal aortic replacement model (n = 30) for 28 days without any antiplatelet therapy. At the termination, all grafts were examined by Doppler ultrasound and then evaluated with histology and scanning electron microscopy. RESULTS: The cumulative release amount of heparin (6.93 ± 1.03 mg) and VEGF165 (22.17 ± 5.56 µg) during 28 days were measured. Endothelial cells cultured on electrospun grafts showed significantly higher attachment efficiency and proliferation compared to the ePTFE ones (P < 0.001). At 2 h more ECs had attached to the P(LLA-CL)/Collagen/Elatin grafts (83.26 ± 8.02%) compared to P(LLA-CL) (67.07 ± 4.16%) and ePTFE (46.87 ± 8.85%). ECs proliferated faster on VEGF loaded grafts (O.D = 2.9 ± 1.2, n = 12) compared to ePTFE (O.D = 1.7 ± 1.0, n = 12). The patency was significantly higher in electrospun grafts (86.6%) than ePTFE grafts (40.0%) (P = 0.021). Correspondingly, the microscope images of electrospun implants showed little thrombus when compared with the ePTFE implants. CONCLUSION: Biofunctionalized electrospun graft showed surgical properties, hemocompatibility and higher short-term patency compared with the ePTFE grafts. Despite good early performances, profound study should be designed for long-term evaluation.

Survival and Functionality of hESC-Derived Retinal Pigment Epithelium Cells Cultured as a Monolayer on Polymer Substrates Transplanted in RCS Rats.

PURPOSE: To determine the safety, survival, and functionality of human embryonic stem cell-derived RPE (hESC-RPE) cells seeded on a polymeric substrate (rCPCB-RPE1 implant) and implanted into the subretinal (SR) space of Royal College of Surgeons (RCS) rats. METHODS: Monolayers of hESC-RPE cells cultured on parylene membrane were transplanted into the SR space of 4-week-old RCS rats. Group 1 (n = 46) received vitronectin-coated parylene membrane without cells (rMSPM+VN), group 2 (n = 59) received rCPCB-RPE1 implants, and group 3 (n = 13) served as the control group. Animals that are selected based on optical coherence tomography screening were subjected to visual function assays using optokinetic (OKN) testing and superior colliculus (SC) electrophysiology. At approximately 25 weeks of age (21 weeks after surgery), the eyes were examined histologically for cell survival, phagocytosis, and local toxicity. RESULTS: Eighty-seven percent of the rCPCB-RPE1-implanted animals showed hESC-RPE survivability. Significant numbers of outer nuclear layer cells were rescued in both group 1 (rMSPM+VN) and group 2 (rCPCB-RPE1) animals. A significantly higher ratio of rod photoreceptor cells to cone photoreceptor cells was found in the rCPCB-RPE1-implanted group. Animals with rCPCB-RPE1 implant showed hESC-RPE cells containing rhodopsin-positive particles in immunohistochemistry, suggesting phagocytic function. Superior colliculus mapping data demonstrated that a significantly higher number of SC sites responded to light stimulus at a lower luminance threshold level in the rCPCB-RPE1-implanted group. Optokinetic data suggested both implantation groups showed improved visual acuity. CONCLUSIONS: These results demonstrate the safety, survival, and functionality of the hESC-RPE monolayer transplantation in an RPE dysfunction rat model.

An Innovative Surgical Technique for Subretinal Transplantation of Human Embryonic Stem Cell-Derived Retinal Pigmented Epithelium in Yucatan Mini Pigs: Preliminary Results.

BACKGROUND AND OBJECTIVE: To develop a safe and efficient surgical procedure for subretinal implantation into porcine eyes of a human embryonic stem cell-derived retinal pigmented epithelium (hESC-RPE) monolayer seeded onto a Parylene-C scaffold. This implant is referred to as CPCB-RPE1. MATERIALS AND METHODS: Ultrathin Parylene-C scaffolds were seeded with hESC-RPE and surgically implanted into the subretinal space of Yucatan mini pigs (n = 8). The surgery consisted of pars plana vitrectomy, induction of a limited retinal detachment, and peripheral retinotomy for insertion of the monolayer using a novel tissue injector, followed by silicone oil tamponade injection, laser photocoagulation around the retinotomy site, and inferior iridectomy. Oral cyclosporine was administered from day 1 and during the entire follow-up period. Three months later, the animals were euthanized and the eyes and major organs were submitted for histological analysis. Adjacent sections underwent immunohistochemical analysis to detect human cells using anti-TRA-1-85 (human blood group antigen) antibody and DAPI antibodies. RESULTS: The cell monolayer was immunopositive for TRA-1-85 3 months after implantation and migration from the Parylene-C scaffold was not detected. One eye had a mild inflammatory reaction around the implant that was negative for human biomarkers. No intraocular or systemic tumors were detected. CONCLUSION: The hESC-RPE cells survived for 3 months in this animal model. The surgical procedure for subretinal implantation of CPCB-RPE1 is feasible and safe, without cell migration off the scaffold or development of ocular or systemic tumors.