WOUND D DR DRES ESSI SING A FAST ST HEA EALING ING PR PROJEC ECT
WOUND D DR DRES ESSI SING A FAST ST HEA EALING ING PR PROJEC ECT OUTLINE INE Intr troducti oduction to Wound nd Dr Dressin ing Mechan anism isms Transition ition Betwee een n Layer ers Bacteria rial Commun unic icati tion on Testing ing Modell llin ing Conc nclusio lusion Referen ence ces s & A Acknow owle ledgeme ement nt
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)
WOUND ND DRES ESSI SING NG : : A FAST ST HEA EALING NG PR PROJEC ECT Which Proteins Will We Use? hEGF : Composed of 53 amino acids and 3 disulfide bonds Increases nucleic acid amount of cell Stimulates cell growth Contributes to developing keratin formation Cause accumulation of glucoseamine and glucosaminoglycan 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 Has no direct effect on cell differentiation Effective on outside wounds
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 osmolytic lysis on target cells Cause opening of new pores on target cells, by creating formation of multimers
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 Lard1 and TliATag is used for signal tag for ABC transport system Helps exportation of proteins containing disulfide bonds Exports proteins with proper folding by exocytosis Functional in E. Coli( taken from Erwinia chrysanthemi) 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? Using both gelatin layer and gelatin microspheres as protein carriers Embedding synthesized granulysin proteins to gelatin sponge Takes place in both inflammatory phase and biosafety 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 What Are the Mechanisms of Wound Dressing? COMMUNICATION MECHANISM 1: Transition Between Layers: Thick Polyurethane Layer Appropriate Media Thin Polyurethane Layer Gelatin Sponge
WOUND ND DRESSING NG : A FAST HEALING NG PROJECT CT How Do We Provide Communication Between Layers? AI-2 Oxygen AHL
WOUND ND DRES ESSI SING NG : : A FAST ST HEA EALING NG PR PROJEC ECT What Are the Mechanisms of Wound Dressing? COMMUNICATION MECHANISM 2: Communication between bacteria colonies:
WOUND ND DRESSING NG : A FAST HEALING NG PROJECT CT What Are Our Devices? Double Double EGF+ pOxygen RBS tetR Terminal pTetR RBS RBS GFP Terminal LARD1 Double Double pCl_lam KGF+ Terminal Terminal pQrr4 RBS RBS LuxI RBS mRFP1 RBS Cl_lam LARD1 Double pLuxR LacI+pl RBS LuxR Terminal RBS LYSIS
Communication between constructed colonies AHL AHL LuxR LuxR
WOUND ND DRES ESSI SING NG : : A FAST ST HEA EALING NG PR PROJEC ECT How Do We Test Our Materials? Oxygen Promoter Protein Transport Gelatin Sponge
WOUND ND DRES ESSI SING NG : : A FAST ST HEA EALING NG PR PROJEC ECT How Do We Test The Oxygen Promoter Activity? 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) VHb gene activity via growth rate 0,71 0,68 0,65 0,62 Optical Density at 600 nm 0,59 Vhb in aerobic cond. 0,56 0,53 0,5 Vhb in anaerobic cond. 0,47 0,44 0,41 E.coli AmpR in aerobic 0,38 cond. 0,35 E.coli AmpR in anaerobic 0,32 0,29 cond. 0,26 0,23 0,2 1st hour 2nd hour 3rd hour 4th hour 5th hour
WOUND ND DRES ESSI SING NG : : A FAST ST HEA EALING NG PR PROJEC ECT How Do We Test Our Materials? Oxygen Promoter Protein Transport Gelatin Sponge
WOUND ND DRES ESSI SING NG : : A FAST ST HEA EALING NG PR PROJEC ECT 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? Thermostable lipase (TliA) is secreted through the ABC transporter. Lipase activity was measured spectrophotometrically using p-nitrophenyl palmitate (pNPP) as a substrate
WOUND ND DRES ESSI SING NG : : A FAST ST HEA EALING NG PR PROJEC ECT How Do We Test TliA and ABC transport mechanism? Synthesized EGF+TliA wtih ABC and without ABC TliA+ABC TliA 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
WOUND ND DRES ESSI SING NG : : A FAST ST HEA EALING NG PR PROJEC ECT How Do We Test Our Materials? hEGF hEGF Gelatin Sponge Oxygen Promoter
WOUND ND DRES ESSI SING NG : : A FAST ST HEA EALING NG PR PROJEC ECT Why Do We Choose Gelatin Instead of Collagen? Gelatin Collagen Difficult to prepare native collagen solutions (1 month period) High absorptive capacity of gelatin compared to collagen For collagen scaffolds, the degree of crosslinking was much lower than that of gelatin
WOUND ND DRES ESSI SING NG : : A FAST ST HEA EALING NG PR PROJEC ECT Why Did We Choose Genipin as crosslinking material? 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 :
WOUND ND DRES ESSI SING NG : : A FAST ST HEA EALING NG PR PROJEC ECT Modelling of Biomaterials Water-binding capacity : The water uptake of the sponges is calculated using the following equation: Release kine netic ics :To determine the possible release mechanism, drug release from collagen sponges was fitted to the following power model:
WOUND ND DRES ESSI SING NG : : A FAST ST HEA EALING NG PR PROJEC ECT What Are Our Future Implications? 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
WOUND ND DRES ESSI SING NG : : A FAST ST HEA EALING NG PR PROJEC ECT How Do We Provide Safety Belt? On/off Switch Mechanism of bacteria via Quorum Sensing Lyophilized bacteria Granulysin embedded gelatin sponge Thin polyurethane layer Thick polyurethane layer Parafilm covering
WOUND ND DRES ESSI SING NG : : A FAST ST HEA EALING NG PR PROJEC ECT Who Helped Us? 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
WOUND ND DRES ESSI SING NG : : A FAST ST HEA EALING NG PR PROJEC ECT Who Helped Us? Sponsors:
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