Polyhydroxyalkanoate-based thin films : characterization and optimization for calcium phosphate crystallization

Abstract

Novel polymer-inorganic composites attract scientific and commercial attention as potential biomaterials for orthopedic applications, due to the fact that currently used materials have still many drawbacks, e.g. problems with cell attachment or degradation products toxicity. Furthermore, scientific research progressively focuses on mimicking the structure and function of the body’s organs. For example, bone is a natural composite of an organic matrix (collagen) and inorganic crystals (calcium phosphate). Such a combination of two components, which alone have disadvantages like poor load bearing of collagen and brittleness of calcium phosphate, enables bone to accept high load and fulfill its functions in the body. Thus, by combining components with complementary properties, materials with improved or novel properties could be produced. In tissue engineering, such materials are then processed into three-dimensional (3D) structural supports, scaffolds for cells, which can be seeded either before implantation to the patient or the patient’s body may serves as a ‘bioreactor’. Most of the currently used scaffolds have been prepared via top-down strategies, using for example bulk materials with additives or by blending inorganic and organic components. Since any foreign material introduced to the body is recognized by its surface, tissue engineering research turns towards controlled assembly of inorganic components at the nanoscale, directed by molecularly organized polymer scaffolds, by a so-called bottom-up approach. One advantage of this strategy is the control and tuning of the scaffold’s surface properties, which enhance the interfacial compatibility between the implant and cells, and consequently may decrease the probability of the implant rejection. Among others, polyhydroxyalkanoates (PHAs), natural microbial polyesters, are very interesting candidates for biomedical applications, due to their biocompatibility and biodegradability. However, PHA-based materials reported so far have been prepared using top-down strategies, without detailed analysis and control of the polymer surface properties. The aim of this thesis was to prepare PHA-based composite materials with potential future applications in orthopedic applications, using the bottom-up approach and to understand i) the scaffold formation, ii) its interactions with most abundant cell membrane components (phospholipids), and iii) templating mechanisms for calcium phosphate crystallization. Poly([R]-3-hydroxy-10-undecenoate) (PHUE), a representative of medium-chain-length PHAs, was investigated. Due to its elastomeric properties the polymer can form a flexible matrix for calcium phosphate crystals, similarly to collagen in bone. PHUE scaffolds were prepared using the Langmuir monolayer technique, which enabled a control over the polymer molecular organization, and produced stable two-dimensional (2D) films on the air-water interface. Interactions of the polymer with biologically important molecules, cell membrane lipids, in mixed Langmuir films were evaluated – this approach is a simple method to model the behavior of living cells in the presence of a synthetic (implant) material. The interactions were highly reliant on the lipid head group size and orientation at the free water surface, and are interpreted considering intra- and intermolecular forces between lipid and polymer molecules. The organic-inorganic composite materials were obtained by using one-component (polymer or lipid) and mixed (polymer-lipid) monomolecular films as templates influencing the growth of calcium phosphate. Crystal size and size distribution, morphology, and composition depend on the nature of organic film-forming molecules and interactions between them. Organic-inorganic composite materials with various properties were achieved by using different lipids and lipid/polymer ratios in the films, and the crystal growth conditions (mineralization time, ions concentration). Briefly, good control of calcium phosphate crystallization was achieved with films containing negatively charged lipid and higher excess of the lipid (for anionic and zwitterionic lipids). This thesis presents the first thorough analysis of PHAs surface properties, which may be helpful to better understand already used PHA-based biomaterials. The study of PHA interactions with lipids provides additional insights for development of e.g. polymer-lipid coating materials. Last, but not least, calcium phosphate crystallization beneath PHAs and its mixed films with lipids may inspire new developments in bone tissue engineering using naturally synthesized polymers. In the broader context, the outcome of this work may have impact not only on PHA-based materials, but also on the understanding of other polyester-based biomaterials. Furthermore, the results may be also of interest for applications where properties of thin, molecularly organized films are crucial for the product design and performance, such as sensors.