HPG Axis .


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HPG Axis. Target Cells. H ypothalamus. -. -. GnRH. LH & FSH. Testosterone. +. Anterior P ituitary. -. -. LH. FSH. Testosterone. Inhibin. +. +. Testosterone. Male G onads. Sertoli Cells. Leydig Cells. Hypogonadism. Hypogonadism
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HPG Axis Target Cells H ypothalamus - GnRH LH & FSH Testosterone + Anterior P ituitary - LH FSH Testosterone Inhibin + Testosterone Male G onads Sertoli Cells Leydig Cells

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Hypogonadism General: Reduction or loss of gonad capacity Target work: Testosterone generation by leydig cells found in male gonads Approach: Restore steroidogenic capacity of leydig cells

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Challenges with customary cell transplantation Immune reaction Foreign body response Advantages of microencapsulation Cell entanglement Immunoisolation Selective transportation Sustained arrival of hormones from ensnared cells Reduced dissemination remove Cell Transplantation

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Microcapsule Parameters Degradation Size rejection by means of work size LH, FSH, O 2 , Nutrients Antibodies Testosterone, Wastes Biocompatibility Microcapsule Size

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O PEGdA O n O Polyethylene glycol (PEG) Synthetic polymer Systematically factor work measure Non-biodegradable Sustained cell insurance Bio-dormant Difficult for cells & proteins to follow Pre-cursor Solution: 10% PEGdA MW12000 0.05% I2959 PBS diluent ± cell suspension PEGdA macromers Photopolymerization (365nm UV light) Polymerization & cross-connecting through free-radical component Swelling PEGdA hydrogel H 2 O

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Problem Design Statement To examine the impacts of hydrogel thickness on the suitability of human prostate disease cells installed inside a polyethylene glycol diacrylate hydrogel. Furthermore, to survey the polymerization and cross-connecting marvels of PEGdA macromers and the diffusive conduct of progesterone through a PEGdA hydrogel lattice. The general objective of this venture is to plan an embodiment framework that offers proficient immunoprotection and powerful dissemination of oxygen, supplements, hormones, and metabolic squanders. This framework, alongside installed human prostate growth cells, will empower the rebuilding of un-controlled hormone levels regularly saw in older folks, and retard the manifestations of maturing.

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Previous Work Used container size of 100µm breadth Observed cell suitability out to 7 days and recognized insignificant testosterone discharge 15 min of UV presentation = edge for maintained cell feasibility Current approach for upgrades Microcapsule measure UV introduction time

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UV Exposure Time versus Level of Hydrogel cross-connecting 14.5 minutes of UV presentation is adequate for cross connecting 3D Swelling Ratio = 3.8

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Thickness ( µm ) 0.0% 0 50 100 150 200 250 - 10.0% - 20.0% - 30.0% Concentration Percent Change in Oxygen - 40.0% - 50.0% - 60.0% Percent Change in Oxygen Concentration at Various Hydrogel Thicknesses as Compared to the Oxygen Concentration at the Site of Implantation Capsule Diameter Post-Swell Testing Range = 25µm ~ 250µm

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Tape spacers Microscope slides PEGdA Hydrogel Sigmacote Preset thickness Hydrogel sandwich Simulation of case span Sigmacote surface treatment to help PEGdA expulsion Post-swell thickness = 25  m ~ 250  m Pre-swell thickness = 25  m ~ 175  m

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Ultrasound Confirmation of swelling count Determine pre/post swell thickness of hydrogel sandwich Transducer Water D PEGdA Microscope Slide Distance (D) = (1/2) x [Time x Speed of Sound]

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Ultrasound Result Linear swollen proportion is 1.54

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Progesterone Diffusion

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Progesterone Diffusion Observed Progesterone discharge over the long run High progesterone levels following 5 hours Progesterone level surpassed straight scope of adjusted bend Data changeability Sex hormone equipped for diffusing out of PEGdA system

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Cell Viability Results Statistical Analysis: 2-test t-test α = 0.05 * Cell Titer-Blue TM Cell Viability Assay * Denotes critical drop from day 2 to day 3 * Denotes noteworthy drop from day 3 to day 4

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Cell Viability Discussion Further information is expected to build up an important pattern and understanding Fluorescence readings near that of the negative control (cell culture medium) Increase number of cells per well or potentially increment brooding time to 3 or 4 hours

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Overall Conclusions PEGdA reasonable material for cell embodiment Sub-deadly UV time necessity @ 14.5 min The work estimate accomplished takes into account the dissemination of progesterone Need to broaden cell reasonability considers for more solid translation

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Future Work Continue to survey hydrogel thickness impact on cell suitability (augmented studies) Evaluate impacts of gel thickness on hormone discharge 2-D & 3-D investigations of the impacts of RGD cell attachment peptides on cell work Fabrication of small scale circles of indicated breadth In vivo examination of exemplification framework

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References ALPCO Diagnostics (2004). Progesterone EIA: For the direct quantitative assurance of Progesterone by protein Immunoassay in human serum. 11-PROGH-305 Version 4.0 Cruise, G. M., Hegre, O. D., Scharp, D. S., & Hubbell, J. A. (1998). An affectability investigation of the key parameters in the interfacial photopolymerization of poly(ethylene glycol) diacrylate upon porcine islets. Biotechnology and bioengineering, 57 (6), 655-665. Journey, G. M., Scharp, D. S., & Hubbell, J. A. (1998). Portrayal of porousness and system structure of interfacially photopolymerized poly(ethylene glycol) diacrylate hydrogels. Biomaterials, 19 (14), 1287-1294. Diramio, J. A., Kisaalita, W. S., Majetich, G. F., & Shimkus, J. M. (2005). Poly(ethylene glycol) methacrylate/dimethacrylate hydrogels for controlled arrival of hydrophobic medications. Biotechnology advance, 21 (4), 1281-1288. Kizilel, S., Perez-Luna, V. H., & Teymour, F. (2004). Photopolymerization of poly(ethylene glycol) diacrylate on eosin- functionalized surfaces. Langmuir : the ACS diary of surfaces and colloids, (20), 8652-8658. Kizilel, S., Sawardecker, E., Teymour, F., & Perez-Luna, V. H. (2006). Consecutive development of covalently fortified hydrogel multilayers through surface started photopolymerization. Biomaterials, 27 (8), 1209-1215. Martens, P. J., Bryant, S. J., & Anseth, K. S. (2003). Fitting the debasement of hydrogels framed from multivinyl poly(ethylene glycol) and poly(vinyl liquor) macromers for ligament tissue designing. Biomacromolecules, 4 (2), 283-292. Mellott. M, Searcy. K, Pishko. M (2001). Arrival of protein from very cross-connected hydrogels of poly(ethylene glycol) diacrylate created by UV polymerization. Biomaterials 22(9):929-41. Muschler. G, Nakamoto C, Griffth L (2004). Building Principles of Clinical Cell-Based Tissue Engineering. The Journal of Bone and Joint Surgery (American) 86:1541-1558 Nuttelman, C. R., Tripodi, M. C., & Anseth, K. S. (2005). Engineered hydrogel specialties that advance hMSC suitability. Lattice biology : diary of the International Society for Matrix Biology, 24 (3), 208-218. Yang. F, Williams. C, Wang. D, Lee. H (2004) The impact of consolidating RGD cement peptide in polyethylene glycol diacrylate hydrogel on osteogenesis of bone marrow stromal cells. Biomaterials. 2005 Oct;26(30):5991-8.

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Special Thanks Chemistry Department Dr. Daesung Lee Yi-Jin Kim VA clinic Yi-Jin Kim Dr. Craig Atwood Miguel Gallego Andrea Wilson Ryan Haasl Promega Corporation Lydia Hwang for her fundamental gift of venture assets CS Hyde Company School of Medicine and Public Health, Medical Physics Dr. Tim Stiles for his assistance in ultrasound estimations Pharmacy Department Dr. John Kao Graduate understudy Amy Chung for her interminable liberality Biomedical Engineering Department Dr. Kristyn Masters and lab Dr. William Murphy and lab Dr. Brenda Ogle and lab

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Micro Albert Kwansa Eric Lee

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encaps John Harrison Miguel Benson Client: Dr. Craig Atwood Advisor: Professor William Murphy

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ulation Yik Ning Wong

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