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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
HPG Axis

Target Cells

Hypothalamus

-

-

GnRH

LH & FSH

Testosterone

+

Anterior Pituitary

-

-

LH

FSH

Testosterone

Inhibin

+

+

Testosterone

Male Gonads

Sertoli

Cells

Leydig

Cells

slide2

Hypogonadism

  • 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
slide3
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

Reduced diffusion distance

Cell Transplantation

microcapsule parameters
Microcapsule Parameters

Degradation

Size exclusion via mesh size

LH, FSH, O2, Nutrients

Antibodies

Testosterone, Wastes

Biocompatibility

Microcapsule Size

polyethylene glycol peg

O

PEGdA

O

O

n

O

Polyethylene glycol (PEG)
  • Synthetic polymer
    • Systematically variable mesh size
  • Non-biodegradable
    • Sustained cell protection
  • Bio-inert
    • Difficult for cells & proteins to adhere

Pre-cursor Solution:

10% PEGdA MW12000

0.05% I2959

PBS diluent

± cell suspension

PEGdA macromers

Photopolymerization

(365nm UV light)

Polymerization & cross-linking via free-radical mechanism

Swelling

PEGdA hydrogel

H2O

problem design statement
Problem Design Statement

To investigate the effects of hydrogel thickness on the viability of human prostate cancer cells embedded within a polyethylene glycol diacrylate hydrogel. Additionally, to assess the polymerization and cross-linking phenomena of PEGdA macromers and the diffusive behavior of progesterone through a PEGdA hydrogel matrix.

The overall goal of this project is to design an encapsulation system that offers efficient immunoprotection and effective diffusion of oxygen, nutrients, hormones, and metabolic wastes.

This system, along with embedded human prostate cancer cells, will enable the restoration of un-regulated hormone levels commonly observed in elders, and retard the symptoms of aging.

slide7

Previous Work

  • Used capsule size of 100µm diameter
  • Observed cell viability out to 7 days and detected negligible testosterone release
  • 15 min of UV exposure = threshold for sustained cell viability
  • Current approach for improvements
    • Microcapsule size
    • UV exposure time
uv exposure time vs degree of hydrogel cross linking
UV Exposure Time vs. Degree of Hydrogel cross-linking

14.5 minutes of UV exposure is sufficient for cross linking

3D Swelling Ratio = 3.8

slide9

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
hydrogel sandwich

Tape spacers

Microscope slides

PEGdA Hydrogel

Sigmacote

Preset thickness

Hydrogel sandwich
  • Simulation of capsule radius
  • Sigmacote surface treatment to aid PEGdA removal
  • Post-swell thickness = 25m ~ 250m
  • Pre-swell thickness = 25m ~ 175m
ultrasound
Ultrasound
  • Confirmation of swelling calculation
  • Determine pre/post swell thickness of hydrogel sandwich

Transducer

Water

D

PEGdA

Microscope Slide

Distance (D) = (1/2) x [Time x Speed of Sound]

ultrasound result
Ultrasound Result
  • Linear swollen ratio is 1.54
progesterone diffusion1
Progesterone Diffusion
  • Observed Progesterone release over time
  • High progesterone levels after 5 hours
  • Progesterone level exceeded linear range of calibrated curve
    • Data variability
  • Sex hormone capable of diffusing out of PEGdA network
cell viability results
Cell Viability Results

Statistical Analysis:

2-sample t-test α = 0.05

*

*

*

*

*

*

*

Cell Titer-BlueTM Cell Viability Assay

* Denotes significant drop from day 2 to day 3

* Denotes significant drop from day 3 to day 4

cell viability discussion
Cell Viability Discussion
  • Further data is needed to establish a meaningful trend and interpretation
  • Fluorescence readings close to that of the negative control (cell culture medium)
    • Increase number of cells per well and/or increase incubation time to 3 or 4 hours
overall conclusions
Overall Conclusions
  • PEGdA suitable material for cell encapsulation
    • Sub-lethal UV time requirement @ 14.5 min
    • The mesh size achieved allows for the diffusion of progesterone
  • Need to extend cell viability studies for more concrete interpretation
future work
Future Work
  • Continue to assess hydrogel thickness effect on cell viability (extended studies)
  • Evaluate effects of gel thickness on hormone release
  • 2-D & 3-D studies of the effects of RGD cell adhesion peptides on cell function
  • Fabrication of micro-spheres of specified diameter
  • In vivo analysis of encapsulation system
references
References
  • ALPCO Diagnostics (2004). Progesterone EIA: For the direct quantitative determination of Progesterone by enzyme Immunoassay in human serum.11-PROGH-305 Version 4.0
  • Cruise, G. M., Hegre, O. D., Scharp, D. S., & Hubbell, J. A. (1998). A sensitivity study of the key parameters in the interfacial photopolymerization of poly(ethylene glycol) diacrylate upon porcine islets. Biotechnology and bioengineering, 57(6), 655-665.
  • Cruise, G. M., Scharp, D. S., & Hubbell, J. A. (1998). Characterization of permeability and network 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 release of hydrophobic drugs. Biotechnology progress, 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 journal of surfaces and colloids, 20(20), 8652-8658.
  • Kizilel, S., Sawardecker, E., Teymour, F., & Perez-Luna, V. H. (2006). Sequential formation of covalently bonded hydrogel multilayers through surface initiated photopolymerization. Biomaterials, 27(8), 1209-1215.
  • Martens, P. J., Bryant, S. J., & Anseth, K. S. (2003). Tailoring the degradation of hydrogels formed from multivinyl poly(ethylene glycol) and poly(vinyl alcohol) macromers for cartilage tissue engineering. Biomacromolecules, 4(2), 283-292.
  • 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
  • Nuttelman, C. R., Tripodi, M. C., & Anseth, K. S. (2005). Synthetic hydrogel niches that promote hMSC viability. Matrix biology : journal of the International Society for Matrix Biology, 24(3), 208-218.
  • 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.
special thanks
Special Thanks
  • Chemistry Department
    • Dr. Daesung Lee
    • Yi-Jin Kim
  • VA hospital
    • Yi-Jin Kim
    • Dr. Craig Atwood
    • Miguel Gallego
    • Andrea Wilson
    • Ryan Haasl
  • Promega Corporation
    • Lydia Hwang for her vital donation of project resources
  • CS Hyde Company
  • School of Medicine and Public Health, Medical Physics
    • Dr. Tim Stiles for his help in ultrasound measurements
  • Pharmacy Department
    • Dr. John Kao
    • Graduate student Amy Chung for her endless generosity
  • Biomedical Engineering Department
    • Dr. Kristyn Masters and lab
    • Dr. William Murphy and lab
    • Dr. Brenda Ogle and lab
micro
Micro

Albert Kwansa Eric Lee

encaps
encaps

John Harrison Miguel Benson

Client: Dr. Craig Atwood

Advisor: Professor William Murphy

ulation
ulation

Yik Ning Wong