WOUND D DR DRES ESSI SING A FAST ST HEA EALING ING PR PROJEC - - PowerPoint PPT Presentation

WOUND D DR DRES ESSI SING A FAST ST HEA EALING ING PR PROJEC ECT

OUTLINE INE

Intr troducti

• duction to Wound

nd Dr Dressin ing Mechan anism isms Transition ition Betwee een n Layer ers Bacteria rial Commun unic icati tion

• n

Testing ing Modell llin ing Conc nclusio lusion Referen ence ces s & A Acknow

• wle

ledgeme ement nt

WOUND D DR DRES ESSI SING A FAST ST HEA EALING ING PR PROJEC ECT

WOUND ND DRES ESSI SING NG : : A FAST ST HEA EALING NG PR PROJEC ECT

Why Do We Need Wound Dressing?

For illnesses which cannot be treated easily ( Burns, diabetes, ulsers like decubitus and venous statis)

Which Proteins Will We Use?

hEGF :

Composed of 53 amino acids and 3 disulfide bonds Increases nucleic acid amount of cell Cause accumulation of glucoseamine and glucosaminoglycan Contributes to developing keratin formation Stimulates cell growth Proven to be synthesized in Escherichia coli

WOUND ND DRES ESSI SING NG : : A FAST ST HEA EALING NG PR PROJEC ECT

Which Proteins Will We Use?

hKGF :

Composed of 194 amino acids and 3 disulfide bonds Acts in regeneration of epithelian tissue Stimulates keratinocyte and epithelian cell division Effective on outside wounds Has no direct effect on cell differentiation

WOUND ND DRES ESSI SING NG : : A FAST ST HEA EALING NG PR PROJEC ECT

Which Proteins Will We Use?

Granulysin :

Composed of 145 amino acids Secreted by cytotoxic T cells Has antimicrobial and antitumor activity Cause opening of new pores on target cells, by creating formation of multimers Cause osmolytic lysis on target cells

WOUND ND DRES ESSI SING NG : : A FAST ST HEA EALING NG PR PROJEC ECT

How Will We Export Our Proteins?

Lard1, TliA and ABC transport Mechanism

Helps exportation of proteins containing disulfide bonds Functional in E. Coli( taken from Erwinia chrysanthemi) Exports proteins with proper folding by exocytosis Lard1 and TliATag is used for signal tag for ABC transport system Uses ATP for transportation

WOUND ND DRES ESSI SING NG : : A FAST ST HEA EALING NG PR PROJEC ECT

How Do We Use Granulysin Protein?

Embedding synthesized granulysin proteins to gelatin sponge Takes place in both inflammatory phase and biosafety Using both gelatin layer and gelatin microspheres as protein carriers Preventing any invasion from upper layers of wound dressing

WOUND ND DRES ESSI SING NG : : A FAST ST HEA EALING NG PR PROJEC ECT

How Do We Fasten The Healing?

Healing Phases:

Inflammation Phase (2-5 days) :

• Phagocytosis ~ Granulysin

Proliferation Phase (2 days-3th week) :

• Epithelization ~ hEGF

Remodelling Phase (3 weeks–2 years) :

• Migration ~ hKGF

WOUND ND DRES ESSI SING NG : : A FAST ST HEA EALING NG PR PROJEC ECT

COMMUNICATION MECHANISM 1:

Transition Between Layers:

Thick Polyurethane Layer Appropriate Media Thin Polyurethane Layer Gelatin Sponge

What Are the Mechanisms of Wound Dressing?

WOUND ND DRES ESSI SING NG : : A FAST ST HEA EALING NG PR PROJEC ECT

How Do We Provide Communication Between Layers?

WOUND ND DRESSING NG : A FAST HEALING NG PROJECT CT

Oxygen

AI-2 AHL

COMMUNICATION MECHANISM 2:

Communication between bacteria colonies:

What Are the Mechanisms of Wound Dressing?

WOUND ND DRES ESSI SING NG : : A FAST ST HEA EALING NG PR PROJEC ECT

What Are Our Devices?

WOUND ND DRESSING NG : A FAST HEALING NG PROJECT CT

pOxygen RBS tetR Double Terminal pTetR RBS EGF+ LARD1 RBS RBS RBS RBS RBS RBS RBS Double Terminal Double Terminal Double Terminal Double Terminal pQrr4 Cl_lam pCl_lam GFP KGF+ LARD1 mRFP1 LuxI LacI+pl LYSIS LuxR pLuxR

LuxR LuxR

AHL AHL

Communication between constructed colonies

How Do We Test Our Materials?

WOUND ND DRES ESSI SING NG : : A FAST ST HEA EALING NG PR PROJEC ECT

Protein Transport

Oxygen Promoter

Gelatin Sponge

How Do We Test The Oxygen Promoter Activity?

WOUND ND DRES ESSI SING NG : : A FAST ST HEA EALING NG PR PROJEC ECT

0,2 0,23 0,26 0,29 0,32 0,35 0,38 0,41 0,44 0,47 0,5 0,53 0,56 0,59 0,62 0,65 0,68 0,71

1st hour 2nd hour 3rd hour 4th hour 5th hour Optical Density at 600 nm

VHb gene activity via growth rate

Vhb in aerobic cond. Vhb in anaerobic cond. E.coli AmpR in aerobic cond. E.coli AmpR in anaerobic cond.

The promoter has become active when oxygen in the environment lowers to %2 (max) When VHb gene activity is high, the growth rate is slightly reduced (Proven by ANOVA test)

How Do We Test Our Materials?

WOUND ND DRES ESSI SING NG : : A FAST ST HEA EALING NG PR PROJEC ECT

Protein Transport Oxygen Promoter Gelatin Sponge

TliA has glyc ycin ineric rich repeats (GG GGXGX GXD) D) in its C-termin inus, , which appear r in many ABC transp sporter- secreted proteins TliA fused protein ins s were excreted to supern rnatant culture succesfu fully lly by detectin ing lipase se activit vity y with tributyrin yrin

WOUND ND DRES ESSI SING NG : : A FAST ST HEA EALING NG PR PROJEC ECT

How Do We Test TliA and ABC transport mechanism?

WOUND ND DRES ESSI SING NG : : A FAST ST HEA EALING NG PR PROJEC ECT

Lipase activity was measured spectrophotometrically using p-nitrophenyl palmitate (pNPP) as a substrate Thermostable lipase (TliA) is secreted through the ABC transporter.

How Do We Test TliA and ABC transport mechanism?

WOUND ND DRES ESSI SING NG : : A FAST ST HEA EALING NG PR PROJEC ECT

TliA fused Proteins were excreted to supernatant Culture successfully Tlia fused proteins were excreted ten- fold more with ABC transporter PrtDEF of Erwinia chrysanthemi Same results took place lipase activity at size of zone around supernatant on tributyrin mixed agar plate

TliA+ABC TliA

Synthesized EGF+TliA wtih ABC and without ABC

How Do We Test Our Materials?

WOUND ND DRES ESSI SING NG : : A FAST ST HEA EALING NG PR PROJEC ECT

hEGF

Oxygen Promoter

Gelatin Sponge hEGF

Why Do We Choose Gelatin Instead of Collagen?

WOUND ND DRES ESSI SING NG : : A FAST ST HEA EALING NG PR PROJEC ECT

Gelatin Collagen

Difficult to prepare native collagen solutions (1 month period) For collagen scaffolds, the degree of crosslinking was much lower than that of gelatin High absorptive capacity of gelatin compared to collagen

Why Did We Choose Genipin as crosslinking material?

WOUND ND DRES ESSI SING NG : : A FAST ST HEA EALING NG PR PROJEC ECT

Crosslinking material is used for determinining time of degradation of polymer. Genipin was used as crosslinking material for both collagen and gelatin It cause degredation time to be neither early nor late.

The crosslinking degree could then be obtained from the differences between the absorbance values before and after the crosslinking. The equation is as follows :

Modelling of Biomaterials

WOUND ND DRES ESSI SING NG : : A FAST ST HEA EALING NG PR PROJEC ECT

Release kine netic ics :To determine the possible release mechanism, drug release from collagen sponges was fitted to the following power model: Water-binding capacity : The water uptake of the sponges is calculated using the following equation:

What Are Our Future Implications?

WOUND ND DRES ESSI SING NG : : A FAST ST HEA EALING NG PR PROJEC ECT

Testing hEGF, Hkgf and Granulysin proteins in vivo Adding some mechanisms of wound healing such as interleukins and thymosin beta 4 to fast wound healing Testing the wound healing mechanism timeline Testing the whole genetic circuit of EGF synthesizing and KGF synthesizing bacteria colonies Adding genomic integration method to increase yield of proteins via Keith Tyo’s (at MIT) novel genomic integration technique

How Do We Provide Safety Belt?

WOUND ND DRES ESSI SING NG : : A FAST ST HEA EALING NG PR PROJEC ECT

On/off Switch Mechanism of bacteria via Quorum Sensing Lyophilized bacteria Granulysin embedded gelatin sponge Thin polyurethane layer Thick polyurethane layer Parafilm covering

Who Helped Us?

WOUND ND DRES ESSI SING NG : : A FAST ST HEA EALING NG PR PROJEC ECT

Acknowledgement

Firstly, we special thanks to Prof Dr Vasıf HASIRCI and Prof Dr Nesrin HASIRCI for their help in preperation of biomaterial-grade Wound Dressing layers. At the design of ABC transporter domain plasmid and signal tag plasmids for EGF, we appretiate very much to Jung Hoon Ahn from Institute for Gifted Students, Korea Advanced Institute of Science and Technology. At the design of oxygen promoter and its usage techniques, thank you very much to Hikmet Geckil, Fulbright Postdoctoral Researcher at Harvard-MIT Health Sciences and Technology and his assistant Emel Aytan Special thanks to Jeffry Rubin for sending KGF plasmid

Who Helped Us?

WOUND ND DRES ESSI SING NG : : A FAST ST HEA EALING NG PR PROJEC ECT

Sponsors:

WOUND ND DRES ESSI SING NG : : A FAST ST HEA EALING NG PR PROJEC ECT

References

• 1. Ulubayram K., Cakar A.N., Korkusuz P., Ertan C. and Hasirci N. “EGF containing gelatin-

based wound dressings.” Biomaterials, accepted 22 September 2000.

• 2. Samuel E. Lynch, Robert B. Colvin, and Harry N. Antoniades. (1989) “Growth Factors in

Wound Healing: Single and Synergistic Effects on Partial Thickness Porcine Skin Wounds.” Institute of Molecular Biology, Boston, Massachusetts. The American Society for Clinical Investigation, Inc. 0021-9738/89/08/0640/07 Volume 84, p. 640-646

• 3. A-H. Salmanian, A. Gushchin, T. Medvedeva, M. R. Noori-Daloii and N. Domansky.

“Synthesis and expression of the gene for human epidermal growth factor in transgenic potato plants.” Biotechnology Letters (September 1996), Volume 18 No. 9 pp.1095-1098.

• 4. Reshma P Shetty, Drew Endy and Thomas F Knight Jr. “Engineering BioBrick vectors from

BioBrick parts.” Journal of Biological Engineering, published April 14, 2008.

• 5. Knight, T. (2009) “Idempotent Vector Design for Standard Assembly of Biobricks.” MIT

Artificial Intelligence Laboratory.

• 6. Sambrook J. & Russell D. W. “Molecular Cloning: A Laboratory Manual”. Cold Spring

Harbor Laboratory Press, Third Edition. MORE REFERENCES AT METU-GENE WIKI

What Are Your Questions and Suggestions?

WOUND ND DRES ESSI SING NG : : A FAST ST HEA EALING NG PR PROJEC ECT