Thermosensitive Hydrogel for Cytokine Gene Therapy in Diabetic Wound Healing

*Pui-Yan Lee, Zhenhua Li, Erin Cobain,Leaf HuangCenter for Pharmacogenetics and Pittsburgh Tissue Engineering Initiative

INTRODUCTION METHODS

RESULTS

RESULTS (Part II)

CONCLUSIONS

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Chronic wounds in diabetic patients featured with delayed inflammatory response, defective collagen formation and remodeling is partially due to

the depletion of growth factor production.

Histology (wound morphology and collagen formation)

•Sacrifice an unwounded mouse and a mouse of each treatment at days 3, 5, and 14 and perform paraffin sectioning. •Analyze the wounds using Hematoxylin and Eosin Staining (wound morphology) and picrosirius red Staining (collagen formation) Purple dots indicates nuclei. Red indicates the presence of collagen.

•Make full size excisional wounds (area = 7 x 7 mm) in 18 diabetic mice (2 wounds per mouse, wounds are uncovered)

•Treat 6 mice with PEG-PLGA-PEG +TGF- 1, 6 mice with PEG-PLGA-PEG, and 6 mice untreated

Wound closure

•Measure the area of the wounds on days 0, 2, 3, 5, 7, 9, 11, and 14

PEG-PLGA-PEG

-It has been previously found as biocompatible material.

-Plasmid DNA containing the TGF- 1 gene was embedded into the polymer matrix and the DNA is most likely released into the wound bed by diffusion.

-It forms a film at 30oC. It is not easily evaporated and necessarily for reapplication which is different from traditional non-occulsive dressings

REFERENCESCH3O(CH2CH2O)x[(COCH2O)y(COCH(CH3)O)z]OCNH(CH2)6NHCO

PEG-PLGA-Urethane-PLGA-PEG

RESULTS (Part I)

Intact skin Untreated PEG-PLGA-PEG PEG-PLGA-PEG + TGF- 1

HE Staining

day5

wound morphology

Picrosirius Red Staining

day5

collagen formation

PEG-PLGA-PEG PEG-PLGA-PEG + TGF- 1

Day 0

Day 5

•Day 9: all mice treated with PEG- PLGA-PEG + TGF- 1 showed 100% wound closure

•Day 11: all mice treated with PEG-PLGA-PEG showed 100% wound closure

•Day 14: all untreated mice showed 100 % wound closure

• * represents p < 0.05

•Mice treated with PEG-PLGA-PEG + TGF- 1 showed a greater percentage of wound closure at Day 5 than mice treated with PEG-PLGA-PEG

Treatment of diabetic wounds with PEG-PLGA-PEG + TGF- 1 results in accelerated wound healing. When compared to the mice treated with PEG-PLGA-PEG and the untreated mice, the mice treated with the TGF- 1 gene displayed evidence of wound repair at a faster rate. In cellular level, the population of skin cells started decreasing to normal level on day 7 ( data not shown). The mice treated with the gene showed:

•Accelerated wound closure

•Faster epithelium formation

•Formation of more mature collagenIt is also important to note that the mice treated with PEG-PLGA-PEG

alone displayed an improved quality of wound healing when compared to the untreated control. Thus, it can be concluded that PEG-PLGA-PEG is beneficial as a wound dressing.

Clark, Richard A. F. The Molecular and Cellular Biology of Wound Repair 2nd Ed. New York: Plenum Press, 1996.

Ziegler, Thomas R., Pierce, Glenn F., Herndon, David N. Growth Factors and Wound Healing: basic sequence and clinical applications. New York: Springer, 1997.

Jeong, Byeongmoon, Bae,You Han, Kim, Sung Wan. “Biodegradable thermosensitive micelles of PEG-PLGA-PEG triblock copolymers.” Colloids and Surfaces B. 1999: 185-193.

PEG PLGA URETHANE

25º 30º 30º

Coagulation Inflammation

Epithelialization

Granulation tissue

Angiogenesis

Remodeling

TGF-beta 1

TGF-beta 1 is a potent chemoattractant and mitogent of fibroblast. It enhances epithelialization, extracellular matrix synthesis such as collagen and proteoglycans.

n

In our study, we used PEG-PLGA-PEG as a wound dressing and at the same time as a cytokine gene vehicle.

However, the presence of proteases and enzymes results in ineffective growth factor therapy. Gene therapy with growth factors has been used as an alternative approach in treating wounds. The ongoing transcription and translation of the cytokine gene allows relatively prolonged availability of cytokine.

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•Black arrows shows the location of the end of epithelial tongue in H&E staining and mature basket-weave collagen pattern in picrosirius red staining