background - hpg axis
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Background - HPG Axis
Testosterone
Target Cells
FSHMale Gonads
Anterior Pituitary
Hypothalamus
GnRH
+
-
+
Testosterone
LH & FSHTestosterone
--
LH+
LeydigCells
SertoliCells
-
Inhibin
Micro
Albert Kwansa
encaps
Eric Lee John Harrison
Client: Dr. Craig Atwood
Advisor: Professor Murphy
ulation
Yik Ning Wong
Hypogonadism General: Reduction or loss of gonad
function Target function: Testosterone
production by leydig cells found in male gonads
Approach: Restore steroidogenic function of leydig cells
Background – Hypogonadism
Challenges with traditional cell transplantation Immune Response Foreign Body Reaction
Advantages of microencapsulation Cell entrapment Immunoisolation Selective transportation Sustained release of hormones from entrapped
cells Micro-scale capsule size
Cell Transplantation
Microcapsule Parameters
Testosterone,Wastes
LH, FSH, O2, Nutrients
Antibodies
Size exclusion via mesh size
Microcapsule Size
Biocompatibility Degradation
Polyethylene glycol (PEG)
Synthetic polymer Systematically variable mesh size
Non-biodegradable Sustained cell protection
Bio-inert Difficult for cells & proteins to adhere
O
O
O
O
n
PEGdA
O
HHOn
PEG
Used capsule size of 100µm diameter
Observed cell viability out to 8 days and detected negligible testosterone release
Current approach for improvements Microcapsule size UV exposure time Adhesion peptide incorporation
Previous Work
Design PEGdA hydrogels for the encapsulation of Murine Leydig Tumor Cells in an effort to increase cell viability and testosterone secretion.
PEGDA hydrogels must provide immunoprotection and allow effective diffusion of oxygen, nutrients, hormones, and metabolic wastes.
Project Design Statement
Testing Range = 25µm ~ 250µmTissue Implant size = 40µm ~ 200µm
Thickness Parameter
Percent Change in Oxygen Concentration at Various HydrogelThicknesses as Compared to the Oxygen Concentration
at the Site of Implantation
-60.0%
-50.0%
-40.0%
-30.0%
-20.0%
-10.0%
0.0%0 50 100 150 200 250
Thickness (Micrometer)
Perc
en
t C
han
ge in
Oxyg
en
Con
cen
trati
on
Tape Spacers
Liquid PEGdA
Microscope Slide (Base)
Microscope Slide (Top)
Ready for UV Exposure
Thickness Methodology
Cross Linking (Swelling Ratio) Mesh Size Stiffness of the PEGdA Network
Effects of UV Exposure Time on Mesh Size
0.00
0.50
1.00
1.50
2.00
2.50
3.00
10 20 30 40
UV Exposure Time (min)
Mesh
Siz
e (
nm
)
UV Exposure on Mechanical Properties of PEGdA
Fabricate thin gels under different UV times & take digital snapshot
Perfuse w/DI H2O, wait until equilibrium swelling is attained, and take second digital snapshot
Compute change in volume via imaging software
UV & PEGdA Hydrogel Swelling
Reduced Hydrogel
Swollen Hydrogel
Swelling Ratio Methodology
PC3 cell culture in 10% serum media Manual counting via hemacytometer UV Time: 0 to 50 min @ 10 min
intervals Incubation for 18 hours at 37oC Cell Titer-Blue Cell Viability Assay
Fluorescence Normalization of fluorescence to cell
number
UV radiation on Cell Viability
Normalized Fluorescence (560/ 590)/ PC3Cell Number vs. UV Exposure Time
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
0 min 10 min 20 min 30 min 40 min 50 min
UV Exposure Time (365nm)
Flu
ore
scen
ce/
PC
3 C
ell
Nu
mb
er
Cell viability 18 hrs after UV exposure
Expected RGD Results
RGD of different concentration and cells are injected into PEGdA
0 – 2.5 mM RGD
RGD effects on secretion
RGD-PEG
0
400
800
1200
1600
2 24 96 168Time (hr)
Macrophage Density adherence on RGD-PEG
Expected RGD ResultsA
dh
eren
t M
acro
ph
age
Den
sity
Mesh size: 4-5 nm
UV exposure time: <10mins
RGD concentration: <2.5mM
Pilot Study Conclusion
Perform cell viability experiments up to 10 min at smaller increments
Cell counting via PicoGreen DNA Assay
Future Work
Mellott. M, Searcy. K, Pishko. M (2001). Release of protein from highly cross-linked hydrogels of poly(ethylene glycol) diacrylate fabricated by UV polymerization. Biomaterials 22(9):929-41.
Muschler. G, Nakamoto C, Griffth L (2004). Engineering Principles of Clinical Cell-Based Tissue Engineering, The Journal of Bone and Joint Surgery (American) 86:1541-1558
Yang. F, Williams. C, Wang. D, Lee. H (2004) The effect of incorporating RGD adhesive peptide in polyethylene glycol diacrylate hydrogel on osteogenesis of bone marrow stromal cells. Biomaterials. 2005 Oct;26(30):5991-8.
References
Dr. Craig Atwood, VA Hospital Professor William Murphy Professor Kristyn Masters Professor John Kao Dr. Daesung Lee Amy Chung Yi Jin Kim Eun Jin Cho
Acknowledgements
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