(593al) Carbon Nanofibers: Rosette Nanotubes Injectable Scaffolds for Myocardial Application

Xiangling Meng¹, David A. Stout², Linlin Sun², Rachel L. Beigessner³, Hicham Fenniri³and Thomas J. Webster^2,4

¹Department of Chemistry, Brown University, Providence, RI 02912, USA

²School of Engineering, Brown University, Providence, RI 02912, USA

³National Institute for Nanotechnology and Departments of Chemistry and Biomedical Engineering, University of Alberta, 11421 Saskatchewan Drive, Edmonton, AB T6G 2M9, Canada

⁴Department of Orthopaedics, Brown University, Providence, RI 02912, USA

Introduction:

Heart failure is a leading cause of death in the world. The lack of blood supply caused by a sudden obstruction in the major blood vessel leads to damage of the cardiac muscle. The rapid wound healing process forms fibrosis instead of cardiac muscle, which may lead to another myocardial infarction in the future.Therefore, the purpose of this in vitro experiment was to design, fabricate and test the ability of a novel injectable biomimetic and conductive material for the regeneration heart tissue.

Materials and Methods:

Combinations of three different materials were tested here: rosette nanotubes (RNTs), carbon nanofibers (CNFs) and pHEMA-poly (2-hydroxyethyl methacrylate). RNTs are soft nanotubes which self-assemble from a synthetic DNA base analog when placed in aqueous solutions. The great cytocompatibilityproperties of RNTs have been proved in osteoblast and endothelial engineering [1,2]. CNFs are conductive and were used here to increase the conductivity of the composites to more closely emulate the conductivity of heart tissue. CNFs used in this study were modified with –OH and –COOH in order to enhance hydrophilicity properties and their dispersion in solution.pHEMA was chosen here since it has been widely used in drug delivery and orthopedicapplications.

CNFs were well dispersed in RNT and HEMA solutions by sonication. The samples were polymerized in an oven under 75℃for 3h. The samples were washed with water (15min×3) in order to remove the unreacted monomers. Samples were sterilized in 70% and 100% ethanol for 5min and dried over night. Samples were rinsed with PBS three times before cell seeding.

Human cardiomyocytes (Celprogen Inc.)were seeded on the composites at a density of 3000 cells cm⁻²for 1day and 3 day proliferation assaysand 3500 cells cm⁻²for 4h adhesion assaysin Human Cardiomyocyte Complete Media supplemented with 10% fetal bovine serum (FBS) under standard cell culture conditions (humidified, 5% CO₂/95% air environment at 37℃). Live cells were counted using the MTT assay.A scanning electron microscope (SEM, LEO 1530-VP) operating at a 10 kV voltage was used to image the composite. Water contact angles on the surface were analyzed by a drop shape analyzer to determine the hydrophilicity of the composites. 3 μl of deionized water was dropped onto the surfaces. The electrical resistance of the samples was determined using a multimeter by touching the surface with two alligator clips. The resistance was tested dry at room temperature and the probes were 10mm apart. An atomic force microscope in contact mode was used to determine the surface roughness of the samples. A sharp tipped cantilever that was 10-50N/m in stiffness was used here and it was calibrated at 32 N/m every time.An Instron Model 5882 Frame with an extension rate of 5mm/min was used to determine the tensile mechanical properties of composites with different concentrations of CNFs and hydrogels controls. Type IVdog shaped scaffolds were used for tensile tests.

All experiments were performedin triplicate, data were plotted as the mean ± standard error of the mean, and statistical analyses were performed. When data were compared, ANOVA software and a student T-test were used. A p-value of < 0.05 was considered to be significant in this study.

Results and Discussion:

Representative SEM pictures showed that RNTs and CNFs were well dispersed in pHEMA via sonication. An increase of surface roughness was observed in samples with greater amounts of CNFs.CNFs increased the conductivity of composites(from 0 to 1.8×10^-3Ω^-1), while RNTs didn’t play an obvious role in increasing conductivity. The contact angle of the samples also decreased from 51.6° to 38.1°, which means the hydrophilicity increased, when adding more CNFs and RNTs. Tensile stress of composites decreased when the concentration of CNFs increased, which is convenient for injecting the composites into the body. Results of the cardiomyocyteadhesion assays  and proliferation assays demonstrated greater cardiomyocyte density on the composites with greater amounts of CNFs and RNTs.

Conclusions: HRNs and CNFs were successfully used to design a novel biomimetic composite to improve cytocompatibility properties for cardiomyocytes and thus, should be further studied for cardiovascular tissue engineering applications.

Acknowledgements: This study was supported by Hermann Foundation, the National Research Council of Canada, the Natural Science and Engineering Research Council of Canada, and the University of Alberta, the National Science FoundationGraduate Research Fellowship Program for David Stout (NSF #1058262).

References:

[1] Zhang L, Rakotondradany F, Myles AJ, Fenniri H, Webster TJ. Arginine-glycine-aspartic acid modified rosette nanotube–hydrogel composites for bone tissue engineering. Biomaterials 2009;30:1309-20.

[2] Fine E, Zhang L, Fenniri H, Webster TJ. Enhanced endothelial cell functions on rosette nanotube-coated titanium vascular stents. Int J Nanomedicine 2009;4:91-7.