1. PLLA-PEG-TCH-labeled bioactive molecule nanofibers for tissue engineering Haiyun Gao 1,2,3, Jun Chen 1,3, Beth Zhou 1,2,3, Wen Zhong 3 and Malcolm Xing 1,2,4 1.Mechanical Engineering, Faculty of Engineering, 2.Manitoba Institute of Child Health, 3.Textile Science, Faculty of Human Ecology and 4. Biochemistry and Medical Genetics, Faculty of Medicine, University of Manitoba.

2. Nanofibers  Fiber with nanodimensions  an extraordinarily high surface area to volume ratio  tunable optical emission  super paramagnetic behavior  Methods to fabricate nanofibers  Drawing  Template synthesis  Temperature-induced phase separation  Molecular self-assembly  Eletrospinning S G Kumbar et al. Biomed. Mater. 3 (2008)

3. Advantages: Simple instrument Continuous process Cost effective compared to other existing methods Scalable Ability to fabricate fiber diameters from few nm to several microns High efficiency for biomedicine application Electrospinning strategy S G Kumbar et al. Biomed. Mater. 3 (2008)

4. Drug Loading in Electrospun Nanofibers (ENs)  Wide applications for ENs:  Fltration, sensors, military protective clothing, photovoltaic devices, liquid- crystal display (LCD), ultra-light weight space craft materials, super-efficient and functional catalysts  Variety of biomedical applications: carriers for drug/therapeutic agent delivery, wound dressing materials and as porous three dimensional scaffolds for engineering various tissues such as skin, blood vessels, nerve, tendon, bone and cartilage Drug loaded in ENs: tetracycline hydrochloride [TCH]  a model antibiotic For my study: TCH

5. PLLA/PLLA-PEG-NH 2 Electrospinning Nanofibers  Materials:  Poly(L-lactide) (PLLA)  Poly(ethylene glycol) (PEG) with functional group PLLA PEG NH 2 Preparation of solution: 1.NaOH + tetrahydrofuran 2.CH 2 Cl 2 + trifluoroacetic acid (TFA) 3.BTAC (surfactant) PLLA/PLLA-PEG-NH 2 electrospun nanofiabers Blending electrospinning conditions: 1.room temperature 2.voltage of 22 kV 3.flow rate of 7 mL/h 4.distance of 12 cm between the needle tip and the collector

6. TCH-loaded Nanofibers  HOW: emulsion electrospinning 3% w/w of TCH + 7.5% w/w PLLA/PLLA-PEG-NH2 + 5% w/w BTAC Emulsification and electrospinning Surface functionalization of electrospun nanofibers for the immobilization of proteins Model proteins: Two fluorescently tagged bovine serum albumins (BSAs) Red: Rhodamin-BSA Green: FITC-BSA

7. Validity of the Immobilization Method NHS was coupled to the carboxyl groups of surface hydrolyzed ENS, resulting in the formation of an NHS ester, with the amide I band at 1646 cm −1 EGS was conjugated to the amino groups of water vapor-treated ENS to form an amide (1646 cm −1 )

8. TCH-loaded Nanofibers Table 1 Sample names and specifications Morphology of the electrospun nanofibers (Scanning electron microscopy micrograph) H0-1 (641nm) H0-3 (608nm) H3-1 (740nm) H3-3 (780nm) Bar: 2 µm.

9. PLLA/PLLA-PEG-NH 2 nanofibers functionalized with both FITC-BSA and rhodamine-BSA Confocal images of PLLA/PLLA-PEG-NH2 nanofibers functionalized with both FITC-BSA and rhodamine-BSA. (A) Image showing FITC-BSA, (B) image showing rhodamine-BSA, and (C) merged A and B. Bar: 20 µm.

10. In Vitro Release of TCH and Antibiotic Susceptibility Test The encapsulation rate of TCH was 70% for ENSs without BSA conjugation (H3-1) and 30% for ENSs with two conjugated BSAs (H3-3).

11. Antibiotic Susceptibility Test Antibacterial tests of H3-1 and H3-3. (A) Day 1 H3-3 (left) and H3-1 (right); (B) day 2 H3-3 (left) and H3-1 (right); (C) day 3 H3-3 (left) and H3-1 (right); and (D) day 4 H3-1 (H3-3 was discarded). Bar: 5 mm.

12. ENs in Tissue Engineering Tissue engineering? Bioresorbable and biocompatible materials native tissues Mimic /Replicate Langer, R. and J. P. Vacanti (1993). "Tissue Engineering." Science 260(5110): 920-926. In one approach to open system implants, three-dimensional highly porous scaffolds composed of synthetic polymers serve as cell transplant devices.

13. ENs in Tissue Engineering ENsTunable porosity cells exchange metabolites and nutrients with environment maintain cellular functionality aid in the reconstruction of tissues maintaining tailored mechanical properties to protect the wound bed from collapse avoid mechanical mismatch between scaffolds and host tissues

14. Cell adhesion and proliferation on nanofibrous scaffold PDGF-BB/RGDS(promote cell attachment)ENs conjugate Immunofluorescence staining of human dermal fibroblasts on ENs of (A) PDGF and RGDS conjugated and (B) blank. Images were recorded by a confocal microscope. The same magnification was used for both pictures. Bar: 100 µm.

15. Conclusion  Multifunctional ENSs were developed that incorporated the antibacterial agent TCH and were successfully surface functionalized with two different bioactive molecules.  This novel material may have potential applications in wound care and tissue engineering.

16. Acknowledgement comes to the Institute of Textile Science, University of Manitoba, Manitoba Institute of Child Health and all my colleagues.