Self-assembling block copolymers in the nucleation of hydroxyapatite

Abstract

Dental erosion is a pandemic affecting 3.6 billion people in the world. Over 95 % of enamel is composed of calcium hydroxyapatite (HAP). Enamel is acellular, and as a result of our lifestyle and daily wear and tear this HAP is irreparably damaged. Currently the main treatment for this damage is a metal or porcelain filling which loses adhesion over time creating larger dental carries. To develop a method that can regenerate natural enamel would provide an evasive alternative to the current dental methods and help stop the dental erosion pandemic. In nature, the growth and nucleation of HAP during enamel formation is regulated by a protein called amelogenin. The protein has hydrophilic-hydrophobic polarity which allows the protein to assemble and has a phosphinic acid group on serine-16 that is crucial in growth and regulation of HAP. Taking inspiration from this protein, self-assembling block copolymers could be used as a synthetic alternative. The benefit of using synthetic polymers in enamel restoration and erosion prevention is that the promotion of CaP can be tuned by changing the functional groups and size of the polymer easily by using controlled radical polymerisation (CRP). They also provide an inexpensive alternative to using protein induced mineralisation methods. Block copolymers can be formed by CRP. They have been useful in many applications such as drug delivery due their ability to form self-assembled structures such as micelle and cylinders. In previous research on dental erosion the focus has been on the ability of polymers to prevent enamel erosion however their ability to promote HAP growth has not been yet investigated. Here phosphonic and carboxylic acids containing block copolymers have been synthesised through ring opening polymerisation (ROP) and reversible addition fragmentation chain-transfer (RAFT) polymerisation in order to form self-assembled structures that promote HAP growth and prevent erosion. PEG-poly(heptenolactone) and PEG-polycaprolatone-poly(heptenolactone) containing carboxylic acids and phosphonate moieties were synthesised using PEG and PEGpolycaprolatone as initiators. In the RAFT polymerisation , phosphonate monomer di(methacryloyloxy)methyl phosphonate (MAPC1), tert-butyl methacrylate (TBuMA), hydroxyethylmethacrylate (HEMA) and 2(dimethylamino)ethyl methacrylate (DMAE) were polymerised with methyl methacrylate (MMA). The resulting polymers were coupled with PEG through esterification and then further polymerised with a second monomer to give a triblock copolymer. Many of the triblock copolymers had critical micelle concentrations and dispersities which were determined through fluorescence spectroscopy and gel permeation chromatography respectively. The morphology of nine polymers were explored further though transmission electron microscopy (TEM). It was found that PMMA-b-PMAPC1acid-b-PEG, PMAPC1acid-b-PMMA-bPEG, PDMAE-b-PMMA-b-PEG all formed spheres between 25 to 50 nm. PHEMA-b-PMMA-b-PEG formed a mixture of short cylinders (worms) and spheres (20 nm and 35 nm respectively) and PMAPC1-b-PEG, PMAPC1acid-b-PEG and PMAA-b-PMMA-b-PEG did not self-assemble. The ability of block copolymers to promote CaP growth was analysed by dynamic light scattering (DLS) as a preliminary test. Of these polymers, PMMA-b-PMAPC1 acid-b-PEG, PMAPC1acid-bPMMA-b-PEG, PHEMA-b-PMMA-b-PEG and PMAPC1acid-b-PEG promoted the precipitation CaP. PMAPC1acid-b-PMMA-b-PEG, on the other hand, did not. PMMA-b-PMAPC1 acid-b-PEG was taken forward and the morphology of the CaP was view under scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The SEM micrographs showed a ball-like matrix of CaP which was confirmed electron dispersive x-ray spectroscopy (EDS). The TEM micrographs also show the ball-like matrix and electron diffraction was also utilized and showed that these structures are amorphous. The PMAPC1acid-b-PMMA-b-PEG, PHEMA-b-PMMA-b-PEG and PMAPC1acid-b-PEG polymers were taken to investigate their ability to protect enamel against acid erosion. Profilometry was used to measure the loss of enamel and SEM was used to observe changes in the enamel surface. PHEMA-b-PMMA-b-PEG and PMAPC1acid-b-PEG provided an acid resistant covering over the enamel resulting in a reduction in enamel loss, however PMAPC1acid-b-PMMA-b-PEG showed minimal reduction. This work has displayed the potential for block copolymers to be a prospective candidate in enamel restoration. However how the polymer controls CaP precipitation needs to be explored further. In particular, how many phosphonic and hydroxyl groups are needed to promote CaP growth. It would also be essential to determine the stability of the amorphous CaP form with PMMA-b-PMAPC1acid-b-PEG to see if at longer periods, the CaP crystallizes.