Age-related macular degeneration: interventional tissue engineering and predictive modeling of disease progression

Thesis (Ph.D.)--Boston UniversityAge-related macular degeneration (AMD) is the leading cause of irreversible blindness in people over the age of 50. As many as 50 million people are affected by AMD worldwide and prevalence is expected to continue to rise due to an aging population. There are two forms of the disease, dry (geographic atrophy) and wet (choroidal neovascularization), both of which result in retinal degeneration and central vision loss. Although anti-vascular endothelial growth factor therapies are moderately successful at treating the wet form, there are no treatments currently available for the more common dry form. Pharmacological therapies have been extensively explored for the treatment of dry AMD, but have achieved little success because the pathogenesis underlying AMD is unknown and likely varies among patients . Recently, tissue engineering has emerged as a promising approach to restore function by replacing diseased retinal tissue with healthy retinal pigment epithelium (RPE). While AMD-associated vision loss occurs when photoreceptors degenerate, this process arises as a consequence of earlier RPE dysfunction. In the healthy retina, the RPE acts as a critical regulator of the microenvironment for both photoreceptors and the nearby vasculature. However in AMD, the RPE no longer performs these essential homeostatic functions leading to photoreceptor apoptosis and vision loss. This dissertation describes the development and in vitro characterization of a tissue engineering scaffold for RPE delivery as potential treatment for dry AMD. First, a novel microfabrication-based method termed "pore casting" was developed to produce thin scaffolds with highly controlled pore size, shape, and spacing. Next, human RPE were cultured on pore-cast poly(c-caprolactone) (PCL) scaffolds and compared to cells on track-etched polyester, the standard RPE culture substrate. RPE on porous PCL demonstrated enhanced maturation and function compared to track-etched polyester including improved pigmentation, barrier formation, gene expression, growth factor secretion, and phagocytic degradation. Lastly, this study established a patient-specific method for predicting AMD progression using retinal oxygen concentration. This approach differs from current diagnosis techniques because it uses physiologically-relevant mechanisms rather than generalized clinical associations which have little, if any, prognostic value

Development and Demonstration of Adanced Tooling Alloys for Molds and Dies

This report summarizes research results in the project Development and Demonstration of Advanced Tooling Alloys for Molds and Dies. Molds, dies and related tooling are used to manufacture most of the plastic and metal products we use every day. Conventional fabrication of molds and dies involves a multiplicity of machining, benching and heat treatment unit operations. This approach is very expensive and time consuming. Rapid Solidifcation Process (RSP) Tooling is a spray-forming technology tailored for producing molds and dies. The appraoch combines rapid solidifcation processing and net-shape materials processing in a single step. An atomized spray of a tool-forming alloy, typically a tool steel, is deposited onto an easy-to-form tool pattern to replicate the pattern's shape and surface features. By so doing, the approach eliminates many machining operations in conventional mold making, significantly reducing cost, lead time and energy. Moreover, rapid solidification creates unique microstructural features by suppressing carbide precipitation and growth, and creating metastable phases. This can result in unique material properties following heat treatment. Spray-formed and aged tool steel dies have exhibited extended life compared to conventional dies in many forming operations such as forging, extrusion and die casting. RSP Tooling technolocy was commercialized with the formation of RSP Tooling, LLC in Solon, Oh

Controlled, pulsatile release of thermostabilized inactivated polio vaccine from PLGA-based microspheres

Many vaccines, such as the inactivated polio vaccine (IPV), must be administered in several doses for full efficacy. Because patient access is a major challenge for vaccination efforts in developing countries, administering multiple doses per patient is impractical in those areas. Single-administration vaccines would greatly improve efforts to vaccinate populations in Third World countries, and the World Health Organization (WHO) Expanded Program for Immunization describes an ideal vaccine as one that is heat-stable, requires only one shot, and is easy to administer. Although already existing technologies, such as microspheres composed of poly(lactic-co-glycolic acid) (PLGA), are able to encapsulate vaccines and release them over an extended period of time up to several weeks, they are not able to maintain antigen stability over the longer time intervals in vivo. Vaccines such as IPV, however, are known to be unstable at elevated temperature, such as the 37°C environment of the body, as well as in the acidic environment of the degrading PLGA microspheres. Please click Additional Files below to see the full abstract

Identification of a synergistic interaction between endothelial cells and retinal pigment epithelium

Abstract The retinal pigment epithelium located between the neurosensory retina and the choroidal vasculature is critical for the function and maintenance of both the photoreceptors and underlying capillary endothelium. While the trophic role of retinal pigment epithelium on choroidal endothelial cells is well recognized, the existence of a reciprocal regulatory function of endothelial cells on retinal pigment epithelium cells remained to be fully characterized. Using a physiological long‐term co‐culture system, we determined the effect of retinal pigment epithelium‐endothelial cell heterotypic interactions on cell survival, behaviour and matrix deposition. Human retinal pigment epithelium and endothelial cells were cultured on opposite sides of polyester transwells for up to 4 weeks in low serum conditions. Cell viability was quantified using a trypan blue assay. Cellular morphology was evaluated by H&E staining, S.E.M. and immunohistochemistry. Retinal pigment epithelium phagocytic function was examined using a fluorescent bead assay. Gene expression analysis was performed on both retinal pigment epithelium and endothelial cells by quantitative PCR. Quantification of extracellular matrix deposition was performed on decellularized transwells stained for collagen IV, fibronectin and fibrillin. Our results showed that presence of endothelial cells significantly improves retinal pigment epithelium maturation and function as indicated by the induction of visual cycle‐associated genes, accumulation of a Bruch's membrane‐like matrix and increase in retinal pigment epithelium phagocytic activity. Co‐culture conditions led to increased expression of anti‐angiogenic growth factors and receptors in both retinal pigment epithelium and endothelial cells compared to monoculture. Tube‐formation assays confirmed that co‐culture with retinal pigment epithelium significantly decreased the angiogenic phenotype of endothelial cells. These findings provide evidence of critical interdependent interactions between retinal pigment epithelium and endothelial cell involved in the maintenance of retinal homeostasis

Thermostabilization of inactivated polio vaccine in PLGA-based microspheres for pulsatile release

AbstractVaccines are a critical clinical tool in preventing illness and death due to infectious diseases and are regularly administered to children and adults across the globe. In order to obtain full protection from many vaccines, an individual needs to receive multiple doses over the course of months. However, vaccine administration in developing countries is limited by the difficulty in consistently delivering a second or third dose, and some vaccines, including the inactivated polio vaccine (IPV), must be injected more than once for efficacy. In addition, IPV does not remain stable over time at elevated temperatures, such as those it would encounter over time in the body if it were to be injected as a single-administration vaccine. In this manuscript, we describe microspheres composed of poly(lactic-co-glycolic acid) (PLGA) that can encapsulate IPV along with stabilizing excipients and release immunogenic IPV over the course of several weeks. Additionally, pH-sensitive, cationic dopants such as Eudragit E polymer caused clinically relevant amounts of stable IPV release upon degradation of the PLGA matrix. Specifically, IPV was released in two separate bursts, mimicking the delivery of two boluses approximately one month apart. In one of our top formulations, 1.4, 1.1, and 1.2 doses of the IPV serotype 1, 2, and 3, respectively, were released within the first few days from 50mg of particles. During the delayed, second burst, 0.5, 0.8, and 0.6 doses of each serotype, respectively, were released; thus, 50mg of these particles released approximately two clinical doses spaced a month apart. Immunization of rats with the leading microsphere formulation showed more robust and long-lasting humoral immune response compared to a single bolus injection and was statistically non-inferior from two bolus injections spaced 1 month apart. By minimizing the number of administrations of a vaccine, such as IPV, this technology can serve as a tool to aid in the eradication of polio and other infectious diseases for the improvement of global health

Histological confirmation of epizootic ulcerative syndrome in two cyprinid species from Lake Liambezi, Zambezi Region, Namibia

Epizootic ulcerative syndrome (EUS) is a fish disease caused by an infection of the oomycete, Aphanomyces invadans. During a fish biodiversity assessment of Lake Liambezi, Zambezi Region, Namibia, in August 2011, two Barbus haasianus and three Barbus unitaeniatus with circular ulcerative skin lesions were collected. Lake Liambezi receives inflow from the Zambezi, Chobe and Linyanti rivers. The presence of EUS in the two species was confirmed histologically by: a loss of epidermis at the site of the lesion; hyphae visible deep into the muscle layer with an associated granulomatous inflammatory reaction; and muscle fibre degeneration visible with associated penetrating hyphae. This paper provides further histological confirmation of EUS from Lake Liambezi and the first record of the disease in B. haasianus

Microstructure and Mechanical Properties of Spray-Formed H13 Steel Tooling

This paper presents results on the microstructure and hardness of spray-formed H13 (Fe-0.40C-5.00Cr-1.10V-1.30Mo (wt%)) tooling. There is very low porosity in both as-spray formed samples and aged samples. The microstructure in the as-spray-formed sample is characterized by primary carbides, acicular lower bainite, and a small amount of martensite and of retained austenite. Spray formed and aged tooling H13 has higher hardness values than those of H13 in conventional tooling. The experimental results of microstructures and hardness are rationalized on the basis of numerical analysis of cooling during processing of spray-formed tooling

Novel pulsatile-release microparticles for single-injection vaccination

Many controlled release devices are designed to achieve near zero-order release kinetics, however for some applications, such as vaccination, non-continuous or pulsatile release is desired. Such pulsatile release systems may enable the creation of single-injection vaccines that eliminate the need for subsequent booster immunizations by spontaneously releasing antigen at time points that correspond to normal vaccination regimens. This would be especially important in the developing world where a lack of consistent access to healthcare contributes to approximately 1.5 million vaccine-preventable deaths each year.1 Here we present the fabrication and characterization of biodegradable core-shell microparticles that exhibit pulsatile release kinetics due to their unique structure. These particles are produced using a novel fabrication process that combines soft lithography, picoliter dispensing, optical alignment, and a gentle heat-based sintering step to generate microparticles with a biodegradable polymeric shell surrounding an antigen-filled core. By altering the composition (e.g. copolymer ratio or molecular weight) of the poly(lactic-co-glycolic acid) shell, particles can be tuned to release discrete pulses of a model antigen at times ranging from four days to two months. This fabrication method is also compatible with sensitive biologics, such as the inactivated polio virus, which retains \u3e80% of its antigenicity after encapsulation. Further, because the shell of the particle is physically separated from the core, these particles can be filled with any aqueous vaccine solution without affecting release kinetics and be easily scaled via massively parallel fabrication. As a result, these particles have exciting potential as single-injection vaccines that fully mimic the antigen presentation profile of traditional bolus injections administered over the course of months or years. Please click Additional Files below to see the full abstract

The proteome of extracellular vesicles released by clastic cells differs based on their substrate

Extracellular vesicles (EVs) from osteoclasts are important regulators in intercellular communication. Here, we investigated the proteome of EVs from clastic cells plated on plastic (clasts), bone (osteoclasts) and dentin (odontoclasts) by two-dimensional high performance liquid chromatography mass spectrometry seeking differences attributable to distinct mineralized matrices. A total of 1,952 proteins were identified. Of the 500 most abundant proteins in EVs, osteoclast and odontoclast EVs were 83.3% identical, while clasts shared 70.7% of the proteins with osteoclasts and 74.2% of proteins with odontoclasts. For each protein, the differences between the total ion count values were mapped to an expression ratio histogram (Z-score) in order to detect proteins differentially expressed. Stabilin-1 and macrophage mannose receptor-1 were significantly-enriched in EVs from odontoclasts compared with osteoclasts (Z = 2.45, Z = 3.34) and clasts (Z = 13.86, Z = 1.81) and were abundant in odontoclast EVs. Numerous less abundant proteins were differentially-enriched. Subunits of known protein complexes were abundant in clastic EVs, and were present at levels consistent with them being in assembled protein complexes. These included the proteasome, COP1, COP9, the T complex and a novel sub-complex of vacuolar H+ -ATPase (V-ATPase), which included the (pro) renin receptor. The (pro) renin receptor was immunoprecipitated using an anti-E-subunit antibody from detergent-solubilized EVs, supporting the idea that the V-ATPase subunits present were in the same protein complex. We conclude that the protein composition of EVs released by clastic cells changes based on the substrate. Clastic EVs are enriched in various protein complexes including a previously undescribed VATPase sub-complex