NANOSTRUCTURED PLGA MEMBRANE FOR SKIN-TISSUE ENGINEERING APPLICATIONS
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1 NANOSTRUCTURED PLGA MEMBRANE FOR SKIN-TISSUE ENGINEERING APPLICATIONS Z. Karahaliloglu 1, B. Ercan 2, E. B. Denkbaş 3, T. J. Webster 2 1 Hacettepe University, Nanotechnology and Nanomedicine Division, 06800, Beytepe, ANKARA, Turkey 2 Northeastern University, Chemical Engineering Department, 02115, Boston, MA, USA 3 Hacettepe University, Chemistry Department, Biochemistry Division, 06800, Beytepe, ANKARA, Turkey 2013 AIChE Annual Chemical Engineering San Francisco, CA
2 BACKGROUND WOUND HEALING Haemostasis Inflammation Proliferation, Epithelization and Maturation Remodelling of the scar tissue Tocco I.. et al. J. Nano Mat., doi: /2012/714134, (2012)
3 BACKGROUND Poly(lactide-co-glycolide) (PLGA) Copolymer PLA&PGA Used in a wide array of FDA approved therapeutic devices. adjustability of degradation rates good mechanical properties (especially toughness and excellent processibility) biocompatibility Poly lactic-co-glycolic acids (PLGA) resorbable sutures, resorbable surgical clips, and controlled-release implants Pan Z. and Ding J., Interface Focus 2, , (2012)
4 BACKGROUND Nanofiber PLGA Microspheres Nanosurface via NaOH Treatment Membranes Porous scaffolds Nanoparticles Yang Y. et al. J. Appl. Polym. Sci. 109(2), , (2008) Untreated PLGA Park G.E. et al. Biomaterials 26, , (2005) Treated PLGA
5 BACKGROUND Problem Currently wound dressing Risk of infection Scar formation Poor integration with host tissue Hypothesis NaOH-treated nanofeatured PLGA membrane can mimick the nanophase topography of the native skin tissue, enhance the adhesion and proliferation of the human epidermal keratinocytes and dermal fibroblasts. Objective The objective of the present study is to investigate the impact of nanostructured PLGA surface on the cell adhesion and growth of skin cells (fibroblasts and keratinocytes) and bacterial response as a wound dressing material.
6 EXPERIMENTAL 50:50wt% PLA:PGA; kda 5% polymer in 6 ml chloroform Pipette 75 µl dissolved PLGA solution onto glass coverslips Allow to dry overnight at room temperature 60 C Sonication at 40 ºC for 30 min Figure: Preparation of PLGA membranes NaOH treatment at different concentration and exposure times
7 SURFACE CHARACTERIZATION (SEM) Untreated PLGA X10 Untreated PLGA X N-10 min X N-10 min X N-10 min X N-10 min X50 1 N-5 min X10 1 N-5 min X50 Figure: SEM images of PLGA membranes at different NaOH concentrations and exposure times
8 SURFACE CHARACTERIZATION (SEM) 1 N-10 min X10 1 N-10 min X50 1 N-20 min X10 1 N-20 min X50 1 N-15 min X10 1 N-15 min X50 Figure: SEM images of PLGA membranes at different NaOH concentrations and exposure times
9 CONTACT ANGLE EXPERIMENT Untreated PLGA 0.1 N-10 min 0.5 N-10 min 1 N-5 min 1 N-10 min 1 N-15 min Water Contact Angle * Untreated PLGA min min 1 N-5 min 1 N-10 min 1 N-15 min 1 N-20 min 1 N-20 min 0 Figure: Water contact angles on PLGA membranes, Values are mean +/- SEM; n=5; *p<0.005 compared to the pure PLGA membrane.
10
11 4h Keratinocyte Adhesion Results Figure: 4hr human epidermal keratinocyte adhesion on untreated and NaOH-treated PLGA membranes. Cellular adhesion increased on nanofeatured PLGA membranes. Values are mean +/- SEM; n=3. *p<0.005, p<0.05 compared to untreated PLGA membrane. Cell Density (Cells/cm 2 ) * Untreated PLGA 0.1 N-10 min 0.5 N-10 min 1 N-5 min 1 N-10 min 1 N-15 min 1 N-20 min 0
12 3 Day Human Fibroblast Cell Proliferation Figure: Human fibroblast proliferation on untreated and NaOH-treated PLGA membranes. Cellular proliferation increased on nanofeatured PLGA membranes. Values are mean +/- SEM; n=3. *p<0.005, p<0.05 compared to untreated PLGA membrane. Cell Density (Cells/cm 2 ) * Day 1 Day 2 Day 3 0 Untreated PLGA 0.1 N-10 min 0.5 N-10 min 1 N-5 min 1 N-10 min 1 N-15 min 1 N-20 min
13 3 Day Human Keratinocyte Cell Proliferation Figure: Human keratinocyte proliferation on untreated and NaOH-treated PLGA membranes. Cellular proliferation increased on nanofeatured PLGA membranes. Values are mean +/- SEM; n=3. *p<0.005, p<0.05 compared to untreated PLGA membrane. Cell Density (Cells/cm 2 ) * * * * * * Day 1 Day 3 0 Untreated PLGA 0.1 N-10 min 0.5 N-10 min 1 N-5 min 1 N-10 min 1 N-15 min 1 N-20 min
14 48 h Bacterial (S. aureus) Adhesion Figure: S. aureus colonies on untreated and NaOH-treated PLGA membranes after 48 hours Untreated PLGA 0.1 N-10 min 0.5 N-10 min 1N-5 min 1 N-10 min 1 N-15 min 1 N-20 min
15 48 h Bacterial (S. aureus) Adhesion Figure: S. aureus colonies on untreated and NaOH-treated PLGA membranes. Bacterial adhesion decreased on NaOHtreated nanofeatured PLGA membrane with low NaOH concentration. Values are mean +/- SEM; n=3. p<0.05. Bacteria Denisty (Cells/ml) 5.00E E E E E E E E E+04 Untreated PLGA min min 1 N-5 min 1 N-10 min 1 N-15 min 1 N-20 min 5.00E E+00
16 DISCUSSION In literature, various studies indicate that the fibroblasts and keratinocytes are sensitive to nanometer scale roughness. 1,2 Therefore, alkaline treatment method is an attractive technique to design the surfaces of the next generation wound healing materials. Figure: Increased keratinocyte density after 1, 3 and 5 days on nanorough Ti substrates compared to nanotubular and unmodified Ti substrates. Figure: Actin was seen to be well formed in cells on both flat (a) and test (b) substrates, although contractile stress fibres were more often observed in cells on the Figure: Graph showing the average cell areas for 27 nm islands. fibroblasts cultured on the planar controls and the 27 nm high islands. 2 Dalby M.J. et al. Biomaterials 25(1), 77-83, (2004) Figure: SEM images of keratinocytes adhering on the (a) nanotubular, (e) nanorough and (i) unmodified Ti after 4 h of culture. 1 Zile M. et al. Acta Biomater. 6(6), , (2010)
17 CONCLUSION A nano-featured surface morphology was successfully created on the surface of PLGA membranes using NaOH solution. In vitro cell culture results demonstrated that base treatment promotes the adhesion of human dermal fibroblast and epidermal keratinocyte. S. aureus bacterial adhesion test results indicate that a low concentration base treatment reduce the bacterial adhesion on the PLGA membrane. Because of all things, nanofeatured PLGA membrane can be a promising alternative for skin-tissue engineering applications.
18 ACKNOWLEDGMENTS Northeastern and Hacettepe University Prof. Dr. Thomas J. Webster and Prof. Dr. Emir Baki Denkbaş Batur Ercan, Ph.D Eric Taylor and Stanley Chung William Fowle
19 Thanks for your attention!!!
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