Electrospun PCL/P3ANA nanofibers as electroactive-bioactive biomaterial for bone tissue regeneration: synthesis, characterization and cell studies

An electroactive and bioactive nanofiber scaffold which serves both chemical and physical bioactive signals for cells, was fabricated. Electrospun poly(ε-caprolactone)/poly(m-anthranilic acid) (PCL/P3ANA) nanofibers were fabricated by addition of increasing amounts of P3ANA to the PCL solutions. The addition of P3ANA in higher amount resulted a decrease in fiber diameter and high surface roughness and large surface area. The increased surface area of PCL/P3ANA nanofibers serves more available sites and carboxyl groups for biofunctionalization with growth factors. The mechanical properties of PCL/P3ANA nanofibers changed due to P3ANA amount which changed the structural properties of the nanofibers. Electrochemical impedance spectroscopy (EIS) measurements showed that the increase of P3ANA amount in the PCL/P3ANA nanofibers resulted lower charge transfer resistance values which indicates higher conductivity. Higher electroactivity is advantageous in terms of delivering electrical signals to cells. Therefore the PCL/P3ANA nanofiber scaffold containing the highest amount of P3ANA with highest surface area, best mechanical and electrochamical properties was chosen for biofunctionalization and electrical stimulation of cells. PCL/P3ANA nanofiber scaffold was biofunctionalized with covalent protein attachment by using 1-ethyl-3-(dimethyl-aminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) activation process. The activation process was investigated and it was found that 50/50 mM of EDC/NHS was the most effective concentration for the activation of PCL/P3ANA nanofibers. After determination of the suitable nanofiber mat for cell culture studies and the optimum concentrations of EDC/NHS for covalent immobilization, PCL/P3ANA nanofiber scaffold was biofunctionalized with growth factors of bone morphogenetic protein-2 (BMP-2) and RGD peptide for in vitro cells studies. The amounts of covalently immobilized BMP-2 and RGD peptide were determined by bicinchoninic acid (BCA) protein assay and with elemental analyzes of N atoms by Energy-Dispersive X-ray Spectroscopy (EDX). Contact angle measurements showed the changes of the surface properties from hydrophobic to hydrophilic after protein immobilization. The increase in the double layer capacitance and charge transfer values of nanofibers after protein immobilization was showed with EIS. Nanofibers were nontoxic and enable for attachment and growth of Saos-2 cells. The cell proliferation was the highest for the RGD peptide immobilized nanofibers. Cell viability is in correlation with cellular adhesion and structure of the scaffold. Cell morphology was polygonal in shape on the PCL, PCL/P3ANA and PCL/P3ANA-RGD nanofibers is similar to each other. The cells on BMP-2 functionalized nanofibers exhibited osteocyte-like morphology. Alkaline phosphatase (ALP) activity and calcium deposition of Saos-2 cells cultured on cover glass, PCL, PCL/P3ANA, BMP-2/PCL/P3ANA and PCL/P3ANA-RGD nanofibers were investigated. The cells on BMP-2 immobilized nanofibers had the highest ALP activity and the highest amount of calcium deposits among all nanofibers and cover glass, which indicates a higher degree of osteogenesis. The effects of electrical stimulation on the differentiation of Bone Marrow Mesenchymal Stem Cells (BMSCs) into bone feature and the ability of PCL/P3ANA nanofibers to deliver the electrical signals to the cell were investigated. Electrical stimulation was applied to the cells with the electric field (voltage difference per unit distance) of 200 mV/mm, 400 mV/mm and 800 mV/mm at frequency of 0.5 kHz, 1 kHz, 5 kHz and 10 kHz. The viability of cells was the highest when frequency of 1 kHz was applied. At frequency of 0.5 kHz and 1 kHz, the highest viability was observed when cells stimulated with 400 mV/mm. When 800 mV/mm AC voltage at any frequency was applied, cell viability was below 50%. The higher frequencies (5 kHz and 10 kHz) caused a dramatic decrease in cell viability. The cells cultured on the PCL/P3ANA nanofibers under electrical stimulation, proliferated by spreading on the nanofiber mats. At 200 mV/mm and 400 mV/mm voltages BMSCs showed osteocyte-like morphology with adherent-cell type actin extensions. Osteogenic differentiation of BMSCs was investigated by staining for ALP activity and Ca deposits. ALP activity and Ca deposition data exhibited similar trend to the proliferation results. The cells stimulated with voltage of 800 mV/mm exhibited almost no ALP activity or mineralization. The highest mineralization and ALP activity was observed when BMSCs stimulated with 400 mV/mm at 1 kHz. The electrical stimulation data suggested that P3ANA in the nanofiber structure was capable of delivering, interacting and mediating the electrical signaling process within the seeded BMSCs.