(578h) Directed Calcium Deposition by Osteoblasts along Carbon Nanofiber Patterns in Polymers

I. Introduction There has been an increasing interest for self-assembled, functionalized, hybrid carbon nanotube based materials for controlling specific cell interactions. Carbon nanotubes/nanofibers have many advantages in tissue engineering, especially in orthopedics. This is because they are light-weight, strong and, possibly more important, can mimic the nanometer structures of components of bone (such as hydroxyapatite and collagen). Bone has also been shown to regenerative under electrical stimuli, thus conductive nanophase materials (like carbon nanotubes) may play a role in promoting osteoblast (bone-forming cells) activity important for orthopedic applications. Previous studies have shown increased adhesion, viability and deposition of calcium by osteoblasts when cultured on carbon nanotube/nanofiber based materials. However, in all of these studies, non-aligned carbon nanotubes/nanofibers in polymers were investigated. Since long bones in the body are highly anisotropic, the objective of this in vitro study was to align micro-patterns of carbon nanofibers in a polymer matrix and determine subsequent osteoblast functions on these constructs.

II. Materials and Methods Carbon Nanofibers (CNFs) Carbon nanofibers produced by carbon vapor deposition were obtained from Applied Sciences, Inc. (Cedarville, OH). These carbon nanofibers have a polynuclear aromatic hydrocarbon (PAH) layer, which is usually called a pyrolytic layer, formed during the production process. Besides hydrophobic properties, a pyrolytic insulating outer layer has a lower surface energy (approximately 25mJ/m2) compared to pyrolytic-free carbon nanofibers (approximately 200mJ/m2). Only carbon nanofibers with a pyrolytic outer layer were used in the present experiments. The diameter of each carbon nanofiber (CNF) used was 100nm.

Polycarbonate Urethane (PCU) An FDA-approved polycarbonate urethane (PCU, catalog # PC-3575A, Thermedics, MA) was used as the model polymer in this study since it has a high melting temperature (above 200˚C), is FDA approved for implantation, and is non-degradable.

Micro-Patterned CNF Arrays on PCU To construct micro-patterns of CNFs on PCU, a novel imprinting method was developed. For CNF alignment, PCU was melted by chloroform, and was coated on a glass surface. After chloroform evaporation, a Au grid (with 20 µm width spacings) was attached. Dispersed CNFs in ethanol were then placed into the spacings of the grid, the Au grid was removed from the PCU surface, and patterned CNF arrays on PCU were subsequently made.

Osteoblast Adhesion Substrates were sterilized in an autoclave and were exposed to UV light for 24 hours before cell culture. Osteoblasts (CRL-11372, American Type Culture Collection, population numbers 2-5) were cultured on the different substrates under standard cell culture conditions (i.e., a 37 °C, humidified, 5% CO2/95% air environment). Osteoblasts were cultured in Dulbecco's modified eagle medium (DMEM, Gibco), supplemented with 10% fetal bovine serum (FBS, Hyclone) and 1% penicillin/streptomycin (P/S, Hyclone), under standard cell culture conditions. Human osteoblasts were seeded at a density of 2,000 cells/cm2 (sub-confluent) onto each substrate and were incubated under standard cell culture conditions in osteoblast growth media (DMEM, 10% FBS, and 1% P/S) for 2 days. At the end of the time period, non adherent cells were removed by rinsing in PBS while adherent cells were fixed with 4% formaldehyde (Fisher) and were stained with Rhodamine Phalloidin (R415, Molecular Probes) to visualize F-actin filaments and Hoechst dye (33258, Sigma) to visualize the nucleus.

Osteoblast Calcium Phosphate Mineral Deposition To determine calcium phosphate mineral deposition on the micro-aligned CNFs in PCU substrates, osteoblasts were cultured (seeding density: 600,000 cells/cm2) in DMEM supplemented with 10% FBS, 1%P/S, 10mM β-glycerophosphate (Sigma), and 50 µg/ml L-Ascorbic Acid (Sigma) under standard cell conditions for 21 days. Osteoblast growth media was replaced every other day. After that time period, cells were lysed with three freeze-thaw cycles in deionized water to leave only the calcium phosphate crystals deposited by osteoblasts. Energy dispersive spectroscopy (EDS) was used to determine deposited mineral chemistry.

Surface Characterization Images of micro-aligned CNF patterns on PCU were evaluated by fluorescence microscopy (DM IRB, Leica) and Scanning Electron Microscopy (SEM: JSM-840, JEOL). For this purpose, CNF arrays on PCU were mounted using double stick carbon tape and were sputter coated with AuPd prior to imaging at room temperature. Cell images were taken using fluorescence microscopy (DM IRB, Leica) with two different excitation wavelengths (400nm and 550nm) to visualize the cell nucleus and f-actin filaments, respectively.

III. Results and Discussion

Aligned CNF in PCU As expected, fluorescence microscopy results of this study showed highly aligned micro-arrays (20µm) of CNFs successfully patterned onto PCU.

Selective Adhesion of Osteoblasts on Micro-patterns of CNFs on PCU The results of the present study provided evidence of selective osteoblast adhesion and alignment on CNFs compared to PCU. Specifically, more than 80% of the osteoblasts adhered on CNF arrays but less than 20% on the PCU portion of the surface.

Selective Deposition of Calcium Phosphate Minerals on Micro-patterns of CNFs on PCU After 21 days of osteoblast culture, directed deposition of calcium phosphate minerals were observed on CNF compared to PCU micro-patterns using SEM and EDS. Thus, the results of this study showed the ability to mimic the alignment of hydroxyapatite in bone on micro-patterned CNFs on PCU. Moreover, since this occurred on the conductive region of the substrate (CNFs), it is possible that future studies could use applied voltages to further improve osteoblast function. Lastly, since adhesion is a prerequisite for osteoblasts to deposit calcium, it was expected that the preferred attachment of osteoblasts on CNF over PCU regions would translate into preferred calcium phosphate mineral deposition directed on CNF micro-patterns.

IV. Conclusions In conclusion, we observed selective osteoblast adhesion on aligned patterns of carbon nanofibers (CNFs) on a polymer (PCU) matrix. Moreover, we observed enhanced calcium phosphate mineral deposition by osteoblasts along CNF micro-patterns on PCU matrices. These results demonstrated the optimal interactions osteoblasts have with CNFs. Their ability to increase osteoblast function may be used as novel implant nanophase materials in bone tissue engineering. Lastly, these results strongly suggest that CNF micro-patterns in PCU should be further studied for orthopedic applications.

V. Acknowledgements The authors would like to thank the NSF for a Nanoscale Exploratory Research grant. We would also like to thank Purdue University for a N. F. Andrew Fellowship.