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