In biological systems, mineralisation is directed by biological macromolecules with anionic functionalised domains. Some of these macromolecules have been implicated in nucleation of biominerals, whilst others are thought to control the growth of nascent crystals through binding to the crystal surface. The structure of these macromolecules and the anionic groups of the functionalised domains varies widely however, making the mechanism by which binding to the crystal surface occurs and the resultant effect on the crystals difficult to elucidate. The aims of this study were to study the growth of a crystal with importance to medicine, hydroxyapatite, in the presence of model polyelectrolytes with carboxylate, sulphate and phosphate functionality in order to determine possible mechanisms of polyelectrolyte-surface interactions.
A general review of biomineralisation is presented, with an emphasis on its occurrence within mammalian systems and the biomacromolecules present as additives during crystal growth. This thesis highlights the few existing reports that have investigated the growth of hydroxyapatite in the presence of polyelectrolyte additives. Due to the common occurrence of polysaccharides in soluble biomacromolecules present during biomineralisation, a set of polysaccharides with varied functional groups; alginate (Alg), a phosphorylated analogue of alginate (PAlg), and heparin (Hep), were investigated for their effect on the crystal growth of hydroxyapatite. The lack of commercial availability of phosphorylated alginate necessitated the development of a protocol for the modification of alginate with phosphate groups, from a phosphorylation reaction previously applied to cellulose. The effects of the reaction on the phosphorylated alginate, such as the degree of substitution, the reduction in Mw, and the site of phosphorylation, were investigated. Upon phosphorylation alginate was found to be modified predominantly at the M3 site, with some modification observed at M2, G2, G3 and the end groups.
The need for the preparation of synthetic polyelectrolytes with defined numbers of subunits and specific functional groups is also identified. A controlled radical polymerisation using RAFT was developed as a method to polymerise monomers with acidic functionality. Through this technique the controlled polymerisation of the monomers 2-hydroxyethyl acrylate (HEA), 3-sulfopropyl acrylate (SPA) and 2-carboxyethyl acrylate (CEA) was conducted. In addition to these homopolymerisations, the co-polymerisation of HEA and CEA was conducted to yield co-polymers with HEA:CEA ratios of 5:1 and 1:1. Kinetic data suggested that the polymerisation proceeded with a high degree of control over the dispersity. The polysaccharides and polyacrylates were used as models in subsequent experiments to extend the knowledge of binding processes of polyelectrolytes and, by extension, biomacromolecules to the surface of hydroxyapatite, through studies of their effect on crystal growth rates and morphologies. A series of crystal growth protocols from the literature were evaluated for their applicability to the study of hydroxyapatite crystal growth rates and morphology. From these, the constant composition method was adopted to quantify the growth rates, along with a relative rates method developed to support the observed rates, with a high temperature synthesis used to evaluate the effect of additives on HAP morphologies. The protocols were repeated with the presence of the polysaccharide and polyacrylate additives. From these experiments it was found that the affinity of binding of the polyelectrolytes to the surface of hydroxyapatite was the result of multiple factors; the number of subunits, the functional groups present, and also potentially the secondary structure of the polyelectrolyte. Phosphorylated alginate was found to cause the greatest reduction in the growth rate and overall size of hydroxyapatite, possibly due to a strong affinity between the phosphate groups and calcium. Despite having a higher number of subunits than PAlg, Alg was found to have a lower affinity to hydroxyapatite, and it is not clear to what extent the functional groups, or secondary structure of either polysaccharide plays a role in this difference. PSPA was found to have the lowest affinity to hydroxyapatite, possibly due to being composed of the smallest number of subunits of all of the polyelectrolytes evaluated. Again the secondary structure and functional groups are expected to play a role in this difference. Heparin, a polysaccharide with carboxyl and sulphate groups was found to bind selectively to the surface normal to the crystal c-axis, suggesting a similar inhibitory action of proteoglycans in biological systems.