Shaping surface acoustic waves for cardiac tissue engineering

The heart is a non-regenerating organ that gradually suffers a loss of cardiac cells and functionality. Given the scarcity of organ donors and complications in existing medical implantation solutions, it is desired to engineer a three-dimensional architecture to successfully control the cardiac cells in vitro and yield true myocardial structures similar to native heart. This thesis investigates the synthesis of a biocompatible gelatin methacrylate hydrogel to promote growth of cardiac cells using biotechnology methodology: surface acoustic waves, to create cell sheets. Firstly, the synthesis of a photo-crosslinkable gelatin methacrylate (GelMA) hydrogel was investigated with different degree of methacrylation concentration. The porous matrix of the hydrogel should be biocompatible, allow cell-cell interaction and promote cell adhesion for growth through the porous network of matrix. The rheological properties, such as polymer concentration, ultraviolet exposure time, viscosity, elasticity and swelling characteristics of the hydrogel were investigated. In tissue engineering hydrogels have been used for embedding cells to mimic native microenvironments while controlling the mechanical properties. Gelatin methacrylate hydrogels have the advantage of allowing such control of mechanical properties in addition to easy compatibility with Lab-on-a-chip methodologies. Secondly in this thesis, standing surface acoustic waves were used to control the degree of movement of cells in the hydrogel and produce three-dimensional engineered scaffolds to investigate in-vitro studies of cardiac muscle electrophysiology and cardiac tissue engineering therapies for myocardial infarction. The acoustic waves were characterized on a piezoelectric substrate, lithium niobate that was micro-fabricated with slanted-finger interdigitated transducers for to generate waves at multiple wavelengths. This characterization successfully created three-dimensional micro-patterning of cells in the constructs through means of one- and two-dimensional non-invasive forces. The micro-patterning was controlled by tuning different input frequencies that allowed manipulation of the cells spatially without any pre- treatment of cells, hydrogel or substrate. This resulted in a synchronous heartbeat being produced in the hydrogel construct. To complement these mechanical forces, work in dielectrophoresis was conducted centred on a method to pattern micro-particles. Although manipulation of particles were shown, difficulties were encountered concerning the close proximity of particles and hydrogel to the microfabricated electrode arrays, dependence on conductivity of hydrogel and difficult manoeuvrability of scaffold from the surface of electrodes precluded measurements on cardiac cells. In addition, COMSOL Multiphysics software was used to investigate the mechanical and electrical forces theoretically acting on the cells. Thirdly, in this thesis the cardiac electrophysiology was investigated using immunostaining techniques to visualize the growth of sarcomeres and gap junctions that promote cell-cell interaction and excitation-contraction of heart muscles. The physiological response of beating of co-cultured cardiomyocytes and cardiac fibroblasts was observed in a synchronous and simultaneous manner closely mimicking the native cardiac impulses. Further investigations were carried out by mechanically stimulating the cells in the three-dimensional hydrogel using standing surface acoustic waves and comparing with traditional two-dimensional flat surface coated with fibronectin. The electrophysiological responses of the cells under the effect of the mechanical stimulations yielded a higher magnitude of contractility, action potential and calcium transient.

Spatial and temporal measurements using polyoxometalate, enzymatic and biofilm layers on a CMOS 0.35 μm 64 X 64-pixel I.S.F.E.T. array sensor

This thesis presents the achievements and scientific work conducted using a previously designed and fabricated 64 x 64-pixel ion camera with the use of a 0.35 μm CMOS technology. We used an array of Ion Sensitive Field Effect Transistors (ISFETs) to monitor and measure chemical and biochemical reactions in real time. The area of our observation was a 4.2 x 4.3 mm silicon chip while the actual ISFET array covered an area of 715.8 x 715.8 μm consisting of 4096 ISFET pixels in total with a 1 μm separation space among them. The ion sensitive layer, the locus where all reactions took place was a silicon nitride layer, the final top layer of the austriamicrosystems 0.35 μm CMOS technology used. Our final measurements presented an average sensitivity of 30 mV/pH. With the addition of extra layers we were able to monitor a 65 mV voltage difference during our experiments with glucose and hexokinase, whereas a difference of 85 mV was detected for a similar glucose reaction mentioned in literature, and a 55 mV voltage difference while performing photosynthesis experiments with a biofilm made from cyanobacteria, whereas a voltage difference of 33.7 mV was detected as presented in literature for a similar cyanobacterial species using voltamemtric methods for detection. To monitor our experiments PXIe-6358 measurement cards were used and measurements were controlled by LabVIEW software. The chip was packaged and encapsulated using a PGA-100 chip carrier and a two-component commercial epoxy. Printed circuit board (PCB) has also been previously designed to provide interface between the chip and the measurement cards.