Comparison Of Growth Of Human Retinal Pigment Epithelial Cell On Two Prosthetic Replacements Of Bruch’s Membrane

BackgroundAge-Related Macular Degeneration (AMD) is one of the leading causes of blindness in those older than age55in the United States and Western Europe. In China, more and more aged people are suffering from AMD with vision loss while the large population is getting older and people’s health care is improved. According to the clinical manifestation, there are two types of AMD,dry AMD and wet AMD. Many AMD patients, especially wet AMD patients, lost their vision acuity resulted from choroidal neovascularization, Retinal Pigment Epithelium Detachment, vitreous hemorrhage, and eventually gloss of macular scar. The poor visual prognosis in this disease has led to the exploration of new techniques to halt or reverse visual loss including photodynamic therapy (PDT), radiation therapy, macular translocation, and pharmacologic therapy.The advantages of PDT over photocoagulation are as follows:it could be used for CNV in macular, selectively for occult neovascularization without any scotoma and damage to neural retina and choroid. PDT is also approved to use in macular choroidal neovascularization secondary to AMD by the Food and Drug Administration department of U.S.A in2000. Although PDT has become increasingly prevalent, it’s usually used for "classic" CNV that covers50percent of the damage and the occult CNV. PDT’s effect on the patients’ vision is limited. Only14%of patients treated with PDT have an improved visual acuity of one or more lines after2years. Another problem present the large number of CNV recurrences reported after laser photocoagulation, and the unpredictable repetition of treatments in3months intervals in PDT treatment. Vision loss occurred in a small part of patients after PDT treatment. Retinal detachment is also a side effect for PDT. The expense of PDT is expensive; it brings a big economical pressure to patients. Anti-angiogenic therapies have become available over the last years and are primarily used for all subgroups of CNV. It includes interleukin (IL), anacortave TM and anti-VEGF-substances. The first drug approved was Pegaptanib, a28base ribonucleic acid aptamer covalently linked to two branched20kD polyethyleneglycol moieties to bind and block the activity of extracellular VEGF, specifically the165amino acid isoform (VEGF165) that is mainly responsible for ocular CNV. Recent animal experiments and also human trials have shown short term efficacy and good tolerability in CNV-AMD with Bevacizumab, which carries the big advantage of a low cost drug and has therefore rapidly gained worldwide use. The recent introduction of an anti-VEGF antibody (ranibizumab) has shown increased promise with more than35%of patients with classic choroidal neovascularization (CNV) experiencing a15-letter visual improvement at one year. Genentech press release:Phase III study shows Lucentis improved vision compared to Visudyne in patients with wet age-related macular degeneration.The most recent addition to the armamentarium for treating exudative AMD is the use of anti-VEGF agents that are intended to block the biologic activities of VEGF on vascular endothelial cells, which include increasing mitogenesis and vascular permeability during the formation of subretinal neovascular complexes. Because subretinal neovascular membranes are already composed of established vascular networks at the time of diagnosis, most of the benefit from anti-VEGF agents is limited mainly to cessation of plasma leakage from incompetent new vessel walls. Anti-VEGF agents require multiple costly intravitreal injections, which can cause retinal detachment and endophthalmitis. Moreover, it is possible that inhibiting one of the multiple regulatory factors and intracellular signaling pathways for VEGF may not necessarily lead to complete inhibition of angiogenesis, because other angiogenic pathways may still be activated. Systemic absorption of anti-VEGF agents also can interrupt physiologic angiogenesis, especially coronary collateral formation and wound healing. The natural history of patients who develop subfoveal choroidal neovascularization from AMD (i.e., exudative AMD) is poor, and until recently there was no uniformly accepted treatment that led to significant recovery of visual function. Many researchers are exploring the possible effective treatments for AMD.It’s the first priotrity to understand the pathology of AMD before finding an effecitive therapy. The RPE plays a big role in the pathology in AMD. RPE is a cuboidal hexagonal monolayer comprising the outermost layer of the retina. Its apical portion faces the outer segments of PRs, and its basolateral surface interacts with the choriocapillaris. There is no vascular around the photoreceptors, which are supported by choroid capillary. The extracellular space at the apical RPE surface is called the subretinal space; it is filled with the interphotoreceptor matrix (IPM). The RPE projects specialized microvilli into the subretinal space that then appose to PRs via specialized IPM domains, the rod and cone sheaths. Together, RPE microvilli and IPM represent the physical components that maintain retinal adhesion. The infolded basal RPE surface rests on BM, a pentalaminar,1-4-um-thick structure combining the basement membranes of the RPE and the choriocapillaris. The RPE secretes and responds to numerous growth factors and other cytokines, such as Pigment Epithelial-Derived Factor (PEDF) and Vascular Epithelial Growth Factor (VEGF). An equilibrium shift in VEGF and PEDF secretion ratios has been implicated as a possible cause of development of choroidal neovascularization (CNV) in AMD. The RPE plays a key role in maintaining retinal function by assuming a strategic position as the metabolic gatekeeper between photoreceptors (PRs) and the choriocapillaries. The RPE and Bruch’s membrane (BM) suffer cumulative damage over lifetime, which is thought to induce age-related macular degeneration (AMD) in susceptible individuals. Maintenance of RPE is important for the function of retina. In AMD, the RPE cells are dysfunctional which is seen in pigment irregularities with either hyper-or depigmentation often together with drusen which results either in the non-neovascular form with progression to geographic atrophy ("atrophic, dry AMD") or neovascular AMD ("exudative, wet AMD") with PED or/and choriodal neovascularization (CNV) accompanied by exudation and hemorrhage and subsequent cell death and scar formationAMD is believed to be caused by progressive deterioration of RPE, BM, and the choriocapillaris-choroid complex, which consequently leads to subsequent damage of the PR cells. Clinical studies have shown that in exudative AMD most visual loss is the result of the patchy loss of retinal pigment epithelial (RPE cells) and disruption of the RPE photoreceptor interface by the ingrowing subretinal fibrovascular tissue rather than photoreceptor cell death. Recently, with the understanding of molecular mechanism of AMD,some researchers suggested the concept of RPE transplantation.RPE transplantation is expected to work in three different ways:(1) It should halt the disease process;(2) It should maintain or improve visual function for an indefinite time;(3) It should stabilize or improve the condition of already damaged PRs and choriocapillaris.Based on the above, many researchers started human RPE transplantation.The early transplantation of RPE started with allogeneic homologous RPE for AMD, unfortunately the vision acuity is not good as expected after transplantation because of graft rejection. Later transplantation of autologous tissue has been propounded. The outcome is also not satisfied. Review all the studies, most of them use RPE cell suspension or single RPE graft into subretina. After transplantation, the RPE cell suspension and graft couldn’t rest on the damaged Bruch’s membrane, survived by isolated cells clump and couldn’t live with RPE monolayer. However, even the isolated cells could attach, their differentiation is limited. The RPE monolayer could play their role with the polar characteristics, otherwise the distributed RPE cell couldn’t work. Fate of the transplanted human fetal RPE depends on the receptor’s Bruch’s membrane (BM).With all the above combined, it is apparent that BM changes likely play a substantial role in RPE dysfunction with aging and/or AMD. Experimental attempts have been devised to reengineer, i.e."clean up" BM to prepare a better matrix for transplantation (Tezel,2004). Ultimately, however, it may be desirable to entirely replace BM with a prosthetic tissue-engineered BM construct. Many groups have tried to study BM by providing a RPE carrier substrate, which at the same time would serve as an either temporary or permanent BM prosthesis. Having such an arrangement enables delivery of an intact epithelial patch that is simultaneously protected from hostile influences of aged BM. Both biological and artificial substrates have been studied up to date, including crosslinked collagen, Gelatin, Fibrinogen,Lens capsule, Descements membrane and Amniotic membrane. But none of them could be an ideal BM prosthesis. Crossed collagen is not degradable and with poor permeability. Transplanted RPE cell could form a monolayer on Fibrinogen, but being an isolated cell "clump" when transplanted with Fibrinogen into subretina, Although the biological substrates could avoid reject reaction, injuries still exist because of surgery.From the above research for BM prosthetics, it’s necessary to find a BM prosthetics which is similar to BM structure (thickness, fiber arrangement, permeability and biocompatibility), and could maintain RPE monolayer and be delivered into the right place in subretina.A tissue-engineering technique is a promising concept. A very simple example is the use of polyester, i.e.poly-lactic acid(PLLA) and poly-DL-lactic-co-glycolic acid(PLGA) films do provide a good matrix for RPE monolayer formation and their engineerable rigidity would aid surgical manipulation, but their neural retinal toxicity upon degradation (giant cell reaction) rendered them unsuitable in initial subretinal implantation experiments, at the same time, the retina injuries during surgery usually oocured because of their rigidity. All these problems limit the use of polyester as an ideal substrate.In the past ten years Tissue engineering, especially nano-technique has been made to develop biocompatible scaffolds for native extracellular matrix (ECM). Native ECM does far more than just provide a physical support for cells. It also provides a substrate with specific ligands for cell adhesion and migration, and regulates cellular proliferation and function by providing various growth factors. In spite of the amazing diversity of ECM structures caused by different biomacromolecules and the way they are organized, a well-known feature of native ECM is the nanoscaled dimensions of their physical structure. In a typical connective tissue, structural protein fibers such as collagen fibers and elastin fibers have diameters ranging from several ten to several hundred nanometers. The nanoscaled protein fibers entangle with each other to form a nonwoven mesh that provides tensile strength and elasticity for the tissues. Researchers as early as the1960s claimed that nanoscaled features influenced cell behaviors. Cells attach to and organize around fibers with diameters smaller than that of the cells. Nanoscaled surface roughness with dimensions ranging from20to50nm produced by chemical etching on silicon wafers enhanced neural cell adhesion and hydroxylase activity. One study reported that osteoblast adhesion, proliferation, alkaline phosphatase activity, and ECM secretion on carbon nanofibers increased with deceasing fiber diameter in the range of60-200nm, whereas the adhesion of other kinds of cells such as chondrocytes, fibroblasts, and smooth muscle cells was not influenced. It has been supposed that the nanoscaled surface affects the conformation of adsorbed adhesion proteins such as vitronectin, thus affecting the cell behaviors. One important kind of ECM in human body is basement membrane, which is a flexible thin (40-120nm thick) mat that underlies all epithelial or endothelial cell sheets to separate them from the underlying connective tissues. As a basement membrane in the eye, BM plays a big role in maintaining RPE monolayer. BM composed mainly of type IV collagen and laminin nanofibers embedded in heparin sulfate proteoglycan hydrogels. The fibers, pores, ridges, and grooves on the basement membrane are all nanoscaled, from several to more than100nm. As an ideal BM prosthestics substrate, its physical structure and chemistry composition should mimic ECM as far as possible. Electrospun polyamide nanofibers matrix is among the most promising biomaterials for BM because of similar physical structure. We establish a hypothesis---Electrospun nanofibers matrix could be an ideal BM prosthesis which support RPE cell growth and differentiation and will contribute to the success of RPE transplantation in AMD. Although there is some study on polyester as the scaffolds for RPE cell, there is no report on etched pore surface modified polyester as the scaffolds for RPE cell up to date, Our work will compare these two prosthetic replacement of BM, explore the possibility of both as the latent future prosthesis of BM,eventually bring a promising idea for RPE transplantation in AMD. There are three parts in this study:Part1:Comparison of human fetal RPE cell growth on two-prosthestic replacement of Bruch’s membrane:etched pore polyester and electrospun nanofiber membranes. Growth and morphology of the RPE cells were monitored under the phase contrast microscope, and the phenotype was identified by immunofluorescence staining and western blot with antibodies against cytokeratin18, tight junction protein ZO-1and RPE65. Scan electric microscopy is used for comparing the arrangement of both prosthesis. With all these experiment results, it’s expected to provide some information for future use of both prosthesesPart2:Comparison of aged human RPE cells growth on two-prosthestic replacement of Bruch’s membrane:etched pore polyester and electrospun nanofiber membranes. Using the same methods as Part1, further compare the possibility of both substrates as an idea future BM prosthesis.Part3:Histology study for both BM prostheses after transplanted into RCS subretinal space. To assess the biocompatibility of both substrates for possible use in subretinal BM prostheses via preliminary histology study.Results:1.EPN resembles the inner collagenous layer of Bruch membrane.2.Both EPN and PET membranes can maintain differentiation of cultured human fetal RPE in vitro. Compared to adult in situ RPE cells, fetal RPE cultured on both two sides surface of EPN and PET can achieve a comparable phenotype with Ca2+Immunostaining to cytokeratin18, ZO-1,RPE65and western blot confirmed human fetal RPE cell could differentiate on both BM prosthses.3.At10days after Ca2+switch, human fetal RPE cells on both sides of EPN had organized into a lattice pattern and the monolayer is irregular,the changes are very significant on the free surface of EPN. At15days, there is detachment on both free surface and plastic-affixed surface of EPN substrates. No detachment is found on the PET substrates although the RPE monolayer is irregular;4.Aged human RPE cell could be cultured successfully with high seeded and Ca2+switch. They show the similar pattern as human fetal RPE cell on the prostheses. Aged RPE cells have a better differentiation on EPN than PET under high seeded;5.As an ideal BM prosthesis, similar physical structure, fiber arrangement, nano-surface characteristics, good permeability and large,flat surface will contribute to the attachment and differentiation of RPE cell;6.The preliminary safety studies in P21RCS Rats suggest gross tolerance of both EPN and PET in subretinal space.Conclusions:1.Electrospun nanofibers are an intriguing prospect for prosthetic BM replacement because of similarity in structure to native BM;2.Although free nanofiber membranes did not support fetal RPE differentiation longterm, this may in part reflect surface topology differences. The effective differentiation of aged RPE on nanofibers suggests that further study is warranted;3.BM replacement may be an important adjunct for RPE replacement strategies in AMD.