Developing electromigration to enhance calcium phosphate crystallisation for bone and enamel remineralisation

In tissue engineering and regenerative medicine of bone and enamel there is a persistent need for fabricating biomaterials capable of replicating the natural material. This demand stems from fundamental limitations that exists in these biological materials. Bone exhibits a fundamental limitation such that it has a finite ability to self-repair and this becomes more significant with age. Enamel faces the issue of dental caries which is a direct result of acid-induced demineralisation that are resultant from mainly cariogenic pathogens. Therefore, this presents a strong motive for the design and development of biomaterials that inherently express the same attributes as those found in the native tissue. Such biomaterials are highly organised architectures comprised of an inorganic component in the form of hydroxyapatite mineral (majority of enamel composition) and an organic element that is collagen (found mainly in bone). The production of these materials involves intricate biological pathways, making them difficult to mimic synthetically. In literature, several methods have set out to biomimetically produce calcium phosphate, with the transient forms to the thermodynamically stable form, hydroxyapatite, being observed. However, none have utilised a method that enhances the rate of crystallisation by controlling the ions involved using an electric field. This work therefore presents a method - called electromigration - that has not been investigated for calcium phosphate, that enhances the rate of this crystallisation, allowing for an effective form of deposition onto biologically relevant scaffolds. This work initially reports the development of the set-up required to allow for a proof of concept study to be conducted. Here, a double diffusion set-up was utilised which separated individual chambers of separate calcium and phosphate solutions by a 50-nm polycarbonate track-etch membrane. A three-electrode system was implemented that comprised of Pt working (WE) and counter electrodes (CE) and a Ag/AgCl reference electrode (RE). Voltammetry studies revealed an operational window of +0.5 - +0.75V which would allow for a predominantly non-faradaic field. Through a Chronoamperometry (CA) v. Chronopotentiometry (CP) study, it was observed that fixing a potential, as opposed to fixing a current, was more effective due to a more controlled field being produced. A 15-minute chronoamperometric study to inspect a step increase in fixed potential, from +0.6V to +0.7V, was conducted. By comparing to passive diffusion, material characterisation via Scanning Electron Microscopy (SEM) and Energy-Dispersive X-ray Spectroscopy (EDX) revealed progressive tendency towards uniform calcium phosphate film deposition, on the HPO4-cotaining side of the membrane, with increasing fixed potential. As this was the main side of crystallisation, under all cases, the efficient formation of films under a field was indicative of selective enhancement of Ca2+ ion migration. A longer study of fixed +0.7V was carried out and at the 1-hour time-point, Transmission Electron Microscopy (TEM) and Selected-Area Electron Diffraction (SAED) illustrated amorphous homogenous films of calcium phosphate. These were composed of nano-metre sized spherical particles that fuse to form this morphology. Under passive diffusion, the apparent morphology was different as a combination of void-filled films and rod-shaped crystallites were observed. At the 2-hour time-point TEM and SAED confirmed the deposition of films and rods in both types of experiments. These were polycrystalline in nature, with indexing verifying characteristic hydroxyapatite reflections. Focused-Ion Beam SEM (FIB-SEM) analysis on rod growth inside the membrane pores showed a greater extent of precipitation in the pores under passive diffusion than with a field. Furthermore, to understand the versatility of the set-up, the electrodes were inverted to inhibit the crystallisation which was demonstrated to be effective after SEM characterisation on both sides of the membrane displayed predominantly no material deposition. Electrochemical Impedance Spectroscopy (EIS) was used to in situ monitor the ion dynamics and ultimately allowed for modelling of the crystallisation process under a field and passive diffusion. Here, a four-electrode system was used to measure ion transport across a membrane, and this was through the addition a Pt sense electrode (SE) to the current three-electrode system. Through equivalent circuit development, impedance fitting of resultant EIS data and literature values of diffusion coefficients, it was found that using a higher fixed potential, the concentrations in the pore of Ca2+ and HPO42- ions were approximately in the kinetic solubility product range (0.15 - 0.39 mM) and corroborates lack of rod formation at early timepoints. Driving the Ca2+ ions via voltage enables more Ca2+ to arrive at the membrane surface for kinetic product formation. This rate out-runs the dissolution rate in the pore, forming solid predominantly at the surface, where a supersaturation is reached at earlier timepoints. Passive diffusion showed concentrations that were in a slightly higher range (0.73 - 2 mM) indicative of zones were the solubility product concentrations and supersaturation concentrations are reached; dissolution-reprecipitation mechanism is most likely in the zones of solubility product concentrations. To conclude this work, the electromigration technique was implemented to collagen substrates to observe the extent of mineralisation. Initially, individual calcium and phosphate solutions were used and after 15 minutes, a network of small nano-metre sized particles deposited on the phosphate side of the membrane. The control experiment did not show the same extent. Taking this result forward enabled the investigation of a field with a calcium phosphate ion clusters (CPICs) solution that is capable of stabilising metastable amorphous calcium phosphate (ACP) to deposit calcium phosphate material. SEM imaging revealed the use of a field facilitates the migration of these ion clusters by depositing thick calcium phosphate plates on the CPIC-containing side. The other side of the membrane (EtOH-containing) shows effective penetration at 15 minutes compared to passive diffusion. Subsequent FIB milling on the CPIC-containing side of the membrane demonstrated the calcium phosphate plates being embedded into the collagen, highlighting a degree of mineralisation. On the EtOH-containing side, collagen fibrils were seen to experience a degree of extrafibrillar mineralisation. The findings in this thesis therefore present proof of principle of utilising electromigration as a method for enhanced calcium phosphate deposition onto inert and biologically relevant scaffolds. Furthermore, the use of EIS has allowed for fundamental ion dynamic modelling of the crystallisation process under both regimes, allowing for an understanding of the physical chemistry. Such results suggest the technique could be of potential use in a clinical setting for restoring mineral onto bone and enamel. However, further material characterisation work is required alongside further optimisation of the migration technique for better mineralisation onto collagen and other biological materials.