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Tissue-Engineered Solutions for Cell Replacement Therapy in Age-Related Macular Degeneration BIOE689Y Fall 2017 Dr. Martha Wang Mostafa Lotfi, Niloofar Massahi, Shannon Schreiner (Team 4) Physiology of the Eye 2 Physiology of the Eye 3 Physiology of the Eye 4 Adult Macular Degeneration (AMD) • Adult Macular Degeneration (AMD) • Leading cause of blindness in the elderly • Affects 2 million Americans • Progressive damage to the macula - central region of the retina • Retina: light receiving nerve layer in the back of the eye • Important for detail and central vision 5 Adult Macular Degeneration (AMD) • Dry AMD • Non-neovascular • Wet AMD • Neovascular 6 Current Treatments • Dry AMD • Bolus injection • hESC-RPE implant • Wet AMD • Anti-VEGF • Anti-PDGF • Macular translocation • RPE transplant 7 Current Treatments • hESC-RPE Derived • iPSC-RPE Derived • Photoreceptor Replacement 8 Project Aims • Global hypothesis: The delivery of a monolayer of hESC-RPE to the ocular subretinal space via a porous, biodegradable, ultra-thin parylene scaffold, impregnated with an anti-VEGF therapeutic, will repair existing RPE damage while simultaneously preventing future neovascularization. • Specifically: 1. Parylene-C film is an amenable scaffold for hESC-RPE while demonstrating biocompatibility with hESC-RPE, choroidal, and retinal tissues 2. Scaffold degradation rates can be controlled by the physical dimensions of the implant 3. Neovascularization will be suppressed by the delivery of an anti-VEGF therapeutic to the local tissues via the implant • Aims proposed: 1. Investigate implant toxicity i.e. genotoxicity, carcinogenicity, blood response, and cytotoxicity. Reproductive toxicity will not be investigated as AMD patients are past their reproductive window. Implant will also be investigated for any adverse mechanical effects that may result from its subretinal placement. 2. Investigate implant degradation rates by varying the radial dimension, thickness, porosity, and pore size of the implant. 3. Investigate bevacizumab and ranibizumab for in vivo suppression of neovascularization of choroidal layer 9 Research Design & Aims – In Silico 10 Research Design & Aims – In Vitro Phase II A 11 Research Design & Aims – In Vitro Phase II B 12 Research Design & Aims – In Vivo Phase III 13 Research Design & Aims – Anti-VEGF Anti-VEGF Suppression of Choroidal Neovascularization 14 Tissue-Engineered​ ​Solutions​ ​for Cell​ ​Replacement​ ​Therapy​ ​in Age-Related​ ​Macular​ ​Degeneration BIOE689Y​ ​Fall​ ​2017 Dr.​ ​Martha​ ​Wang Mostafa​ ​Lotfi,​ ​Niloofar​ ​Massahi,​ ​Shannon​ ​Schreiner​ ​(Team​ ​4) Fischell​ ​Department​ ​of​ ​Bioengineering “We​ ​pledge​ ​on​ ​our​ ​honor​ ​that​ ​we​ ​have​ ​not​ ​given​ ​or​ ​received​ ​any unauthorized​ ​assistance​ ​in​ ​this​ ​assignment” Abstract According​ ​to​ ​the​ ​World​ ​Health​ ​Organization,​ ​adult​ ​macular​ ​degeneration​ ​(AMD)​ ​ranks​ ​third​ ​among global​ ​causes​ ​of​ ​visual​ ​impairment​ ​with​ ​a​ ​blindness​ ​prevalence​ ​of​ ​8.7%.​ ​AMD​ ​is​ ​the​ ​primary​ ​cause​ ​of visual​ ​deficiency​ ​in​ ​industrialized​ ​countries​​ ​and​ ​affects​ ​2​ ​million​ ​Americans.(Nazari​ ​et​ ​al.)​ ​As​ ​RPE tissue​ ​does​ ​not​ ​have​ ​regenerative​ ​capacity​ ​and​ ​the​ ​root​ ​cause​ ​of​ ​AMD​ ​is​ ​unknown​ ​at​ ​this​ ​time, research​ ​is​ ​focused​ ​on​ ​treatment​ ​strategies​ ​which​ ​include​ ​the​ ​use​ ​of​ ​human​ ​embryonic​ ​stem​ ​cell derived​ ​retinal​ ​pigmented​ ​epithelium​ ​(hESC-RPE).​ ​ ​This​ ​proposal​ ​seeks​ ​to​ ​deliver​ ​a​ ​monolayer​ ​of hESC-RPE​ ​to​ ​the​ ​ocular​ ​subretinal​ ​space​ ​via​ ​porous,​ ​biodegradable,​ ​ultra-thin​ ​parylene​ ​scaffold, impregnated​ ​with​ ​an​ ​anti-VEGF​ ​therapeutic,​ ​to​ ​repair​ ​existing​ ​RPE​ ​damage​ ​while​ ​simultaneously preventing​ ​future​ ​neovascularization.​ ​ ​Implant​ ​viability​ ​will​ ​be​ ​evaluated​ ​based​ ​on​ ​cytotoxicity, including​ ​any​ ​adverse​ ​mechanical​ ​effects​ ​that​ ​may​ ​result​ ​from​ ​its​ ​subretinal​ ​placement,​ ​implant degradation​ ​rates,​ ​and​ ​suppression​ ​of​ ​neovascularization​ ​of​ ​the​ ​choroidal​ ​layer​ ​through​ ​two​ ​potential anti-VEGF​ ​drugs,​ ​bevacizumab​ ​and​ ​ranibizumab. Clinical​ ​Need There are two types of adult macular degeneration (AMD): dry (non-neovascular) and wet (neovascular). Both forms of AMD are characterized by discoloration of the retinal pigment epithelium (RPE), a buildup of cellular debris known as a drusen formation, followed by a thickening of Bruch’s membrane.(Birch and Liang) Early stage indicators and risk factors for the two types of AMD are similar,​ ​but​ ​late​ ​stage​ ​development​ ​and​ ​presentation​ ​are​ ​distinct. Dry AMD is characterized by drusen formations in the back of the eye.(Birch and Liang) Drusen formations in patients with AMD, although present in normal eyes as well, have entered a vicious cycle and accumulated to form larger, more rigid aggregates. (Sarks, Sarks, and Killingsworth) In these regions, drusen eventually fade away and are replaced by a patch of dead RPE termed geographic atrophy (GA).(Nazari et al.) The primary challenge in the treatment of dry AMD is a lack of knowledge of its root cause and although a variety of risk factors have been identified, currently there is no consensus regarding the specific cause of AMD.(Nazari​ ​et​ ​al.) Wet AMD is characterized by the formation of blood vessels below the retina.(Kaarniranta et al.) The barrier function of the new vessels is compromised and results in leakage of plasma to form edematous regions under the retina or into the eye.(Kaarniranta et al.) It is known that inappropriate regulation of vascular endothelial growth factor (VEGF) is responsible for the growth of these new vessels.(Nazari et al.)​1 Management of VEGF is a strategy for slowing down the progression of wet AMD​ ​but​ ​does​ ​not​ ​stop​ ​the​ ​inevitable​ ​result. 1 Current​ ​Strategies​ ​for​ ​Treatment Dry​ ​AMD​ ​Treatment At this time, there are no clinical treatment options for dry AMD as the root cause is unknown (Rickman, C.B., LaVail, M.M., Anderson, R.E., Grimm, C., Hollyfield, J., Ash) however, there are a variety of cell therapy treatments being investigated. Current research is focused on the use of RPE implants obtained from either human embryonic or induced pluripotent stem cells by either bolus injection of RPE cell suspension or via prefabricated RPE stem cell sheets implanted into the subretinal space.(Olmos et al., 2015) According to Vugler et al, in vitro experiments demonstrated hESC-RPE could form a monolayer of regular, polarised cells which demonstrated apical and basal regions and expressed markers of both developing and mature RPE.(Vugler et al.) Additionally, characteristics of RPE cells cultured on collagen membranes have been shown to develop similar differentiation​ ​indicators​ ​to​ ​ ​RPE​ ​cells​ ​in​ ​vivo.(White​ ​and​ ​Olabisi) Experimental evidence indicates hESC-RPE has a survival period of 12 months in immunocompromised laboratory models and a polarized monolayer of hESC-RPE had a higher survival rate than suspensions without a teratoma or ectopic development.(Diniz et al.) This suggests the probability of a positive association of hESC-RPE in human retinal subjects as well. Due to the possibility of implant rejection, immune response needs to be considered, and immunosuppressant medications required for these therapies.(Olmos et al.) Experimental evidence has also indicated favorable survival rates, and functionality of hESC derived RPE in the form of monolayer on polymer substrates (rCPCB-RPE1 cells​/​pluripotent stem cells) in cultured cells in rats.(Thomas et al.; Tian et al.;​ ​Thumann​ ​et​ ​al.) Wet​ ​AMD​ ​Treatment Since it has been shown that VEGF regulation is important in the progression of wet AMD, pharmaceutical companies have begun producing anti-VEGF treatment options. Examples of these anti-VEGF drugs are Pegaptanib (ss nucleic acid), Ranibizumab (monoclonal antibody), and Aflibercept (recombinant fusion protein).(Avery et al.) Stated briefly, all of these treatments work by binding or blocking VEGF-A or a combination of VEGF isomers.(Nazari et al.) All 3 are FDA approved for use in the treatment of wet AMD and require monthly or semi-monthly injections of the agent into the eye.(Avery et al.) The motivation behind these treatments is, if the rate of angiogenesis can be decreased, patients will have a better prognosis; however, this strategy requires early diagnosis. In the same vein as the treatments above, a cursory search of ClinicalTrials.gov revealed there are many co-treatment trials where anti-VEGF therapies have been paired with anti-platelet derived growth​ ​factor​ ​(anti-PDGF)​ ​which​ ​is​ ​important​ ​in​ ​the​ ​formation​ ​of​ ​blood​ ​vessels. 2 Macular translocation and RPE transplantation surgery have also been options for patients suffering from AMD.(Steven D Schwartz et al.) Both of these surgical interventions demonstrate healthy RPE tissue can have vision restorative capabilities which have been demonstrated to last at least 5 years.(Oshima et al.) However, these surgical options can be technically challenging and may result in​ ​vitreoretinopathy​ ​or​ ​torsion.​ ​(Nazari​ ​et​ ​al.) Current​ ​Investigated​ ​Tissue​ ​Engineering​ ​Strategies​ ​for​ ​Treatment hESC-RPE​ ​derived Transplantation of human embryonic stem cell - derived retinal pigment epithelium (hESC-RPE) has been demonstrated as an effective and promising strategy in the treatment of AMD. In a study published in 2016, Schwartz, et al. produced results that indicated it was possible to implant hESC-RPE in patients suffering from dry AMD resulting in restoration of function of both photoreceptors and visual function.(Steven D. Schwartz et al.) ​These conclusions were based primarily on the tolerance and safety of the hESC-RPE cells and secondarily on the efficacy of the transplanted​ ​cells. In this study, 18 patients (9 dry AMD, 9 STGD [Stargardt Disease]) were injected sub-retinally with a target dose of resuspended hESC-RPE. Each group of 9 patients was further subdivided into 3 cohorts, each receiving a differing number of hESC-RPE cells: 50,000, 100,000, or 150,000. The patients were then given a 6 week regimen of immunosuppressants including tacrolimus and mycophenolate mofetil; after the first 6 weeks had elapsed, the tacrolimus was discontinued and mycophenolate mofetil was continued to be administered for an additional 6 weeks. Following the procedure, all patients were serially observed for overall changes in health as well as changes in visual capabilities. To deem the procedure as safe, patients were physically examined, had their vitals taken and underwent electrocardiograms, hematological and serological testing as well as cancer screening. Although no patient in the study suffered an intraocular or systemic event as a result of the hESC cells, a number of patients were observed to have adverse effects as a result of the immunosuppressants​ ​administered. To examine the visual outcomes, the patients were examined for best-corrected visual acuity (BCVA), visual fields, slit-lamp biomicroscopy, ophthalmoscopy, OCT, fluorescein angiography, autofluorescence, fundus photography and electroretinography. Thirteen out of 18 patients exhibited an increase in subretinal pigmentation and at the 6 month mark, 9 out of the 18 eyes demonstrated stable​ ​or​ ​better​ ​visual​ ​performance​ ​on​ ​a​ ​visual​ ​acuity​ ​test. While these results are promising, the use of hESCs is not without risk or controversy. Although hESCs are not sourced from an abortion their use remains contentious. Additionally, the use of hESCs​ ​can​ ​result​ ​in​ ​the​ ​formation​ ​of​ ​a​ ​teratoma,​ ​unwanted​ ​differentiation,​ ​and​ ​immunologic​ ​rejection. iPSC-RPE​ ​derived In 2006, Yamanaka et. al. showed that by overexpressing 4 factors (OCT3/4, Sox2, c-Myc, Klf-4) fibroblasts can be induced into an embryonic-like pluripotent state.(Takahashi and Yamanaka) ​These cells were called induced pluripotent stem cells (iPSC) and are another renewable source of stem cells that can be differentiated into a variety of tissues. Also, because these cells can be generated from readily available fibroblasts, this method opens up the potential for creating autologous transplant where a patient’s own cells can be reverted to the iPSC state, differentiated, and then implanted back into the patient. There have been two primary methods of utilizing iPSC-derived RPE (iPSC-RPE) in a cure for AMD. Both 3 involve the transplantation of iPSC- RPE in the eye at locations where later stage macular degeneration has caused the loss of the RPE layer. The first method is the transplantation of a suspension of RPE cells at the back of the eye.(Westenskow et al.) The second method is the transplantation of a monolayer of iPSC-RPE on a scaffold at the back of the eye. Both methods have drawbacks​ ​and​ ​design​ ​challenges. In the injection of iPSC-RPE suspension, the cells are cultured, differentiated, loaded into syringe, and injected into the eye. There are a few similar culturing methods available in the literature, the details of which are beyond the scope of this paper however, it is necessary to note that in culturing iPSCs the purity of the final cellular product is an omnipresent hurdle regardless of the cell type being produced. In both methods, the integration of the cells into the eye is a concern that needs to be addressed. In the suspension-injection method, integration is optimized by placing the cells at a very specific location in the back of the eye. Unfortunately, this requires expert surgical skills and misplacement is common.(Westenskow​ ​et​ ​al.) In the iPSC-RPE monolayer placement method, a single layer of cultured RPE is grown on a scaffold and placed in the back of the eye.(Bird et al.) Many of the challenges in the injection method are applicable here as well. Purity is a concern, placement can be a hassle, and this method has the added challenge of a scaffold. This method has many advantages over the bolus approach which include: a rigid scaffold for easier placement of the cells in the desired location and the ability to analyze​ ​the​ ​cells​ ​in​ ​their​ ​macro-structural​ ​formation​ ​(as​ ​a​ ​monolayer​ ​sheet). Photoreceptor​ ​replacement There are several transplant strategies that meet the requirement for a successful photoreceptor replacement therapy however, in all cases, donor cells must be able to migrate to the recipient retina and differentiate into a functional and connected photoreceptors.(Westenskow et al.) Whole retinal sheets from embryonic and neonatal retinae have the ability to differentiate. In a study completed by Radtke et al, scientists saw an improvement in vision in 7 out of 10 AMD patients after receiving fetal retinal sheet transplants.(Pearson) Transplantation of brain derived neural stem cells into the neural retina is another possibility that researchers have investigated. However, these integrated cells do not show intrinsic characteristics of mature retinal neurons.(Iyer et al.) ​To overcome this issue stem cells from embryonic retinas can be used as they are able to differentiate into glial cells and cells expressing specific photoreceptors.(Pearson) However, they are not able to integrate into the laminar structure of retina. Successful transplantation also depends on the developmental stage of the donor cell. This was seen in the study completed by MacLaren et al. where postnatal donor cells were transplanted into the recipients at the exact developmental stage. The cells migrated and matured into normal​ ​photoreceptors.(Qiu​ ​et​ ​al.) One of the challenges faced after transplanting photoreceptors is their ability to improve vision. A study completed by Pearson et al suggests that there is a direct correlation between the number of integrated cells and the amount of vision restored.(MacLaren et al.) The researchers also used gene replacement therapy to show that 120,000 functioning photoreceptors are needed to produce scotopic electroretinogram​ ​response.(Pearson​ ​et​ ​al.) The described strategies for photoreceptor replacement are derived from postnatal retina which occurs in the second trimester in humans and therefore supply is limited. Researchers are investigating reproducible sources of photoreceptor precursors. Embryonic stem cells derived from the inner cell mass of a preimplantation blastocyst have also shown great promise in their ability to generate retinal cells. In a study published by Eiraku et al, researchers were able to develop a 3D culture in which the ESCs mimicked retina morphology.(Eiraku et al.) The reprogramming of nuclei of somatic cells, to the pluripotent state, can bypass the problems associated with the use of human embryos. These induced pluripotent stem cells should have similar capability as ESCs; in a study published by Lamba et al, investigators showed that the iPSCs can generate photoreceptor like cells but​ ​can​ ​have​ ​some​ ​resistant​ ​to​ ​retinal​ ​differentiation.(Lamba​ ​et​ ​al.) The realistic prospect of photoreceptor transplantation presents an important landmark in therapies for patients​ ​suffering​ ​from​ ​AMD​ ​as​ ​these​ ​cells​ ​are​ ​able​ ​to​ ​integrate​ ​into​ ​recipient​ ​retina​ ​and​ ​restore​ ​vision. 4 Project​ ​Aims Age related macular degeneration, which stems from a loss or damage to the Retinal Pigmented Epithelium (RPE), is the leading cause of visual disability in the western world.(Idelson et al.; Oshima et al.) ​RPE tissue is characterized by a monolayer of hexagonal cells, which are filled with pigment, and is located between the highly vascularized choroidal layer of the eye and the neuroepithelium, which contains rod and cone photoreceptors.(Maminishkis et al.) ​These hexagonal cells have well defined apical and basolateral regions as well as tight junctions between cells which help to form a blood-retinal barrier.(Peyman, Spitznas, and Straatsma) ​The RPE’s main functions are: phagocytosis of rod and cone outer segment fragments, shed from their distal ends; uptake, processing, transport, and release of vitamin A (retinol); regulation of net ion and water movement in the apical to basal direction; absorption of stray light, thus improving visual images; and to aid in the attachment of the retina to the choroidal layer.(Idelson et al.) ​Derived from the neuroectoderm, RPE, once differentiated, does not self-renew and must remain functional for the lifetime of the individual. (Klimanskaya​ ​et​ ​al.;​ ​Bok) In the eyes of patients with “wet” AMD, neovascularization of the choroidal layer, through Bruch’s membrane and into the subretinal space, leads to leakage of blood and serum, causing a severe decrease in central vision.(Maminishkis et al.) This neovascularization has been attributed to an overabundance of Vascular Endothelial Growth Factor present in the region.(Spilsbury et al.) If the RPE below the fovea suffers sufficient damage, potential medical interventions are few in number and have limited effectiveness in the treatment of this degenerative disease.(Oshima et al.) ​Existing therapeutic treatments of AMD, which include intravitreal injections of anti-vascular growth factor, and triamcinolone, aim only to slow or halt the progression of neovascularization; they lack the ability to reverse the loss of best-corrected visual acuity (BCVA).(Idelson et al.) However, recent developments have​ ​indicated​ ​new​ ​treatment​ ​strategies​ ​are​ ​on​ ​the​ ​horizon. The use of Human Embryonic Stem Cell RPE (Idelson et al.) (hESC-RPE), to replace dysfunctional RPE, has been demonstrated to be a viable method to restore visual function in those with AMD. (Maminishkis et al.; Steven D. Schwartz et al.; Song et al.) Participants of clinical studies have shown stabilization or improvement of visual acuity, ​while simultaneously supporting the safety of the use of hESCs in this application.(Steven D. Schwartz et al.; Song et al.) Therapeutic replacement hESC-RPE can be delivered to the intraretinal space via cell suspension or as cell layer(s) adhered to an implantable material. Multiple studies have demonstrated that the use of a monolayer of hESC-RPE has an outcome superior to that of a cell suspension.(Diniz et al.; Thomas et al.) There is a wide variety of materials to which the hESC-RPE could be adhered.(Montezuma et al.) In studies investigating the intra-retinal implantation safety of parylene-C, a poly(p-xylylene) polymer, outcomes indicate this material demonstrates a low incidence of histological disruption.(Montezuma et al.; Yu et al.; Wang et al.) Once in place, the replacement RPE should be protected from damage associated with neovascularized AMD. Anti-VEGF therapies, such as bevacizumab and ranibizumab, have been shown as equally effective to one another in the prevention of the formation of new vasculature as a treatment for wet AMD.(Martin et al.; Berg et al.; Told et al.) ​In both instances, patients who received anti-VEGF treatments displayed gains in visual acuity, especially when therapeutics were delivered on a​ ​scheduled​ ​versus​ ​as-needed​ ​basis.(Martin​ ​et​ ​al.;​ ​Berg​ ​et​ ​al.;​ ​Told​ ​et​ ​al.) The global hypothesis of this proposed work is the delivery of a monolayer of hESC-RPE to the ocular subretinal space via a porous, biodegradable, ultra-thin parylene scaffold, impregnated with an anti-VEGF therapeutic, will repair existing RPE damage while simultaneously preventing future neovascularization.​ ​Specifically,​ ​we​ ​hypothesize: 1. Parylene-C film is an amenable scaffold for hESC-RPE while demonstrating biocompatibility with​ ​hESC-RPE,​ ​choroidal,​ ​and​ ​retinal​ ​tissues 2. Scaffold​ ​degradation​ ​rates​ ​can​ ​be​ ​controlled​ ​by​ ​the​ ​physical​ ​dimensions​ ​of​ ​the​ ​implant 3. Neovascularization will be suppressed by the delivery of an anti-VEGF therapeutic to the local tissues​ ​via​ ​the​ ​implant 5 To​ ​address​ ​these​ ​hypotheses,​ ​the​ ​following​ ​specific​ ​aims​ ​are​ ​proposed: 1. Investigate implant toxicity i.e. genotoxicity, carcinogenicity, blood response, and cytotoxicity. Reproductive toxicity will not be investigated as AMD patients are past their reproductive window. Implant will also be investigated for any adverse mechanical effects that may result from​ ​its​ ​subretinal​ ​placement. 2. Investigate implant degradation rates by varying the radial dimension, thickness, porosity, and pore​ ​size​ ​of​ ​the​ ​implant. 3. Investigate bevacizumab and ranibizumab for in vivo suppression of neovascularization of choroidal​ ​layer Research​ ​Design​ ​&​ ​Aims In order to treat conditions like AMD, via hESC-RPE, it is essential to verify the efficacy of the different approaches in animal models prior to initiating human trials. A mouse model is an efficient analog for testing the viability of hESC-RPE for potential carcinogenicity, blood pathology, genotoxicity, etc. However, there is a lack of information on the use of these methods in human models and instances of human trials are absent. Moreover, the evidence collected to date indicates potential changes in stem cells as well as their microenvironments. There is also little data regarding the cell-intrinsic stem cell changes, as well as the potential toxicity in human implants.(Piccin and Morshead) Although hESCs are considered pluripotent, they appear to demonstrate a predisposition for specific cell lineage differentiation. An addition, a significant hurdle in the use of hESCs is their potential to form teratomas or other neoplasms; preclinical assessment of this tissue is required to mitigate the long-term tumorigenic potential of hESC-RPE.(Cunningham et al.) The development of proliferative vitreoretinopathy (PVR), macular pucker or macular hole, as well as crust or scar tissue development, can lead to loss of visual acuity due to an implant.(Alexander et al.) Due to these varied risks, all research needs to be validated by experimentation, and in depth analysis conducted, to ascertain a Parylene-C film, coated with a monolayer of hESC-RPE and anti-VEGF growth factors, is an amenable,​ ​biocompatible,​ ​and​ ​effective​ ​scaffold,​ ​which​ ​can​ ​be​ ​used​ ​for​ ​the​ ​treatment​ ​of​ ​AMD. In​ ​Silico Modeling​ ​of​ ​optimal​ ​implant​ ​dimensions​ ​and​ ​degradation​ ​rate Phase I of this research will focus on the theoretical optimization of the dimensions of the implant that will replace the dysfunctional RPE. This investigation will examine how implant degradation rates are affected by varying the radial dimension, thickness, porosity, and pore size of the implant. Determination of the range of the radial dimension is based on documented values obtained for a mouse retinal region, plus or minus 10%. The central value for the radial dimension will be 0.75mm,(Remtulla and Hallett) thus resulting in a modeling radial range of 0.675 – 0.825mm. Ideally, the value to be used for the thickness of the parylene-C implant would be rooted in experimental values obtained for the basement membrane thickness of 54nm.(Cuthbertson and Mandel) However, as this thickness would prove extremely challenging to manufacture, coat with cells, and physically implant in a subject’s eye, the thinnest value of the implant to be modeled will be based on actual material availability of 0.2µm(Parylene Engineering), and previous studies which indicate ranges of values of 0.15-0.30µm approximate the permeability of Bruch’s membranes.(Lu et al.) ​The range of values to be modeled for the thickness parameter will represent a variability of baseline to baseline plus 25% or 0.2 – 0.25µm. The third parameter to be considered in the in silico model is the size of the pores in the parylene-C film. The desired range would be too small to support neovascularization but large enough to easily accommodate the transport of nutrients and waste. The range to be modeled is representative of a manufacturing reproducible minimum porous membrane array (PMA) which has pore diameters of 110nm(Thakar et al.) to 10x this value, or 1100nm (1.1 µm) which is well below the minimum diameter value of rodent or other mammal’s capillaries(Iatridis) of 3µm. Finally, the last parameter to be modeled is the porosity of the scaffold; the optimal value would accommodate the cell density required to have functional RPE while also being sufficient for waste/nutrient transport. Based on the data presented in a study completed in 2002 by Del Priore, Kuo, and Tezel (Del Priore, Kuo, and Tezel), RPE cell density should meet or exceed 4980 ± 90 cells/mm​2​.(Iatridis) With a given hexagonal diameter of 12µm,(Bhatia et al.) each RPE cell would 6 occupy 124.77µm​2​, thus in order to maintain the 4980cells/mm​2 threshold, 0.62135mm​2 of every square millimeter of scaffold would need to be present. Thus, porosities would be modeled for values ranging from 0-35%. The vitreous humor turnover rate will be modeled using values obtained by Laurent and Fraser (Laurent and Fraser) indicating diffusion controlled transport through the vitreous body; rate constants for components with high molecular weights (>500,000) will be set to 0.024/d, mid-range molecular weights (~18 000) to 0.16/d and low molecular weight (
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Tissue-Engineered Solutions for
Cell Replacement Therapy in
Age-Related Macular Degeneration

BIOE689Y Fall 2017
Dr. Martha Wang

Mostafa Lotfi, Niloofar Massahi, Shannon Schreiner (Team 4)

Physiology of the Eye

2

Physiology of the Eye

3

Physiology of the Eye

4

Adult Macular Degeneration (AMD)
• Adult Macular Degeneration (AMD)
• Leading cause of blindness in the elderly
• Affects 2 million Americans
• Progressive damage to the macula - central region of
the retina
• Retina: light receiving nerve layer in the back of the
eye
• Important for detail and central vision

5

Adult Macular Degeneration (AMD)
• Dry AMD
• Non-neovascular

• Wet AMD
• Neovascular

6

Current Treatments
• Dry AMD
• Bolus injection
• hESC-RPE implant
• Wet AMD
• Anti-VEGF
• Anti-PDGF
• Macular translocation
• RPE transplant

7

Current Treatments
• hESC-RPE Derived

• iPSC-RPE Derived
• Photoreceptor Replacement

8

Project Aims
• Global hypothesis:
The delivery of a monolayer of hESC-RPE to the ocular subretinal space via a porous,
biodegradable, ultra-thin parylene scaffold, impregnated with an anti-VEGF therapeutic,
will repair existing RPE damage while simultaneously preventing future neovascularization.

• Specifically:
1. Parylene-C film is an amenable scaffold for hESC-RPE while demonstrating biocompatibility with hESC-RPE,

choroidal, and retinal tissues
2. Scaffold degradation rates can be controlled by the physical dimensions of the implant
3. Neovascularization will be suppressed by the delivery of an anti-VEGF therapeutic to the local tissues via the
implant

• Aims proposed:
1. Investigate implant toxicity i.e. genotoxicity, carcinogenicity, blood response, and cytotoxicity. Reproductive

toxicity will not be in...


Anonymous
Really great stuff, couldn't ask for more.

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