The effect of poling state, surface charge, and frequency of vibration of piezoelectric poly (vinylidene fluoride) films for bone and neural tissue engineering applications

Novel paradigms for tissue engineering recognize the need for active or smart scaffolds in order to properly regenerate specific tissues. Electrical and electromechanical cues are the most relevant in promoting functionality in tissues such as nerve, muscle, and bone, among others. The existence of electrical phenomena within certain tissues may suggest the requirement of such phenomena (ie., electroactivity, piezoelectricity) during tissue regeneration. For instance, it has been shown that electrically charged surfaces can influence different aspects of cell behavior such as growth, adhesion, or morphology of different cell types, including osteoblast, neural, and muscular cells. Therefore, electroactive materials and, in particular, piezoelectric ones, show a strong potential for novel tissue engineering strategies. Piezoelectric materials have an interesting ability to vary surface charge when a mechanical load is applied, without the need for an external power source or connection wires, a feature that can be taken advantage of in novel tissue engineering strategies. Poly (vinylidene fluoride) (PVDF) is a semi-crystalline and biocompatible polymer with the largest piezoelectric response among piezoelectric polymers, mechanical properties appropriate for tissue engineering applications, and excellent electroactive properties such as piezo-, pyro and ferroelectricity. It was hypothesized that by varying vibration frequencies in piezoelectric substrates, attached neuronal cells would respond with varying onsets of growth. Since nerves innervate both bones and muscles, we further hypothesized that frequencies that promoted neural growth would also promote bone and muscle cell growth. The first aim of this study sought to investigate the effect of oscillating electric fields on a variety of mesenchymal tissues—human mesenchymal stem cells (hMSCs), bone (osteoblasts), and nerve cells by seeding them on poled and unpoled PVDF membranes and vibrating them at 20, 60, and 100 Hz. The results of this study indicated significant increases in osteogenic activity for both osteoblasts and hMSCs when subjected to mechanical vibration and the piezoelectric effect. Metabolic activity assays of hMSCs and osteoblasts verified that proliferation of both cell types was enhanced due to the piezoelectric effect of poled PVDF films but reduced in response to mechanical stimulation alone. Neurite imaging of undifferentiated and differentiated nerve cells revealed increases in neurite growth in response to mechanical and electrical stimulation. Bone is itself piezoelectric, it follows that bone cells would respond to piezoelectric substrates. Nerves also come into direct contact with bone, thus it follows that the piezoelectric properties of bone also affect nerve cells. Therefore, the second hypothesis is that piezoelectric substrates with a surface charge most mimicking that of bone will promote increased adhesion and proliferation of bone and nerve cells. Thus, on the second aim of this dissertation is to examine the effect of stationary electric fields on a variety of mesenchymal tissues—human mesenchymal stem cells (hMSCs), bone, and nerve cells by seeding them on tissue culture polystyrene and three kinds of PVDF film surfaces: unpoled films with no surface charge, poled films with cells cultured on the positively charged side of the sample, and poled films with cells cultured on the negatively charged side of the sample. The same methods that were used in investigating the effect of oscillating electric fields on cells were employed to observe how the stationary electric field affects cells differentiation and growth and at the same time points. The results showed a more homogeneous distribution of hMSCs and osteoblasts seeded on negatively poled PVDF films, but no osteogenesis. Metabolic activity assays of hMSCs and osteoblasts indicated that the highest number of viable hMSCs resulted on negatively poled PVDF films while the highest number of viable osteoblasts occurred on positively poled PVDF films. Finally, neurite imaging verified that charged piezoelectric PVDF membranes induce neurite outgrowth more than electrically neutral membranes in the absence of electrical stimulation. The final goal of this study was to fully characterize the dynamics of the loading environment cells were subjected to, which has not been previously reported in PVDF cell studies, and to correlate the measurements to cell fate. Directly measuring PVDF and media displacement permitted calculation of the actual acceleration PVDF and cells were subjected to and illustrated that the cell culture media has a significant impact on the oscillating pressure imparted to the films and thus the piezoelectric output of the PVDF. From these measurements, it was possible to estimate the voltage output of the PVDF films, which for 100 Hz vibrations were in the physiological range of the action potentials that are experienced by excitable cells such as muscle and nerve. These results suggest a cause for the observed change in morphology of hMSCs towards neuronal cells. The results from this study may better define optimal stimulation parameters for desired cell fate and has already resulted in unexpected and new findings not yet reported in the literature.Ph.D.Includes bibliographical referencesby Rabab Chalab