New biomimetic approaches for producing bone-like calcium-phosphate coatings on the surface of tissue engineering 3D architectures and orthopaedic Implants

Bone is one of the most wonderful examples of nature’s ability to engineer living materials. The processes by which the mineralized tissues are formed can be a source of information for the development of new materials for biomedical applications, capable of better mimicking living tissues, i.e. biomimetic materials. In the field of bone replacement and regeneration, this new concept can lead to innovative ideas towards the controlled fabrication of advanced materials. When considering a particular material as being adequate for orthopaedics one should keep always in mind that it must be mechanical and biologically compatible with bone. Furthermore, it may be advantageous to exhibit a bone bonding behaviour, i.e. to express bioactivity by creating an environment compatible with osteogenisis (bone growth), having the mineralizing interface developing as a natural bonding junction between living and non-living materials. This interface can be generated by a calcium phosphate coating at the surface of the material. The techniques whereby Ca-P layers are produced have recently undergone a revolutionary change leading to profound consequences in their potential applications. Over the last years, calcium phosphate layers were deposited upon the surfaces of implants under non-physiological conditions, which limits their application in polymeric substrates. In the recent years, there has been an increasing interest in the so-called biomimetic preparation of calcium phosphate coatings, under physiological conditions of temperature, pH and pressure. This new generation of coating techniques as enlarged the spectrum of potential applications of such coatings from bone fixation improvement in traditional bone replacement applications, to coating of 3D scaffold architectures in Tissue Engineering applications. Due to their processing versatility, these coatings can be tailored in terms of chemical composition, crystallinity and resorbability or even be loaded with bioactive molecules and/or serve as scaffolds for the seeding of living cells, stimulating bone formation. To do this on a degradable polymer is not a very easy task, as the surface of the substrate (and pH around it) is continuously changing. These were the main challenges addressed in this thesis, having always in mind that to date scientists did not produce yet any material capable of mimicking the bone structure. In this way nature is, and will continue to be, the best material scientist, and the only one able to design complex structures and control intricate processing routes that lead to the final shape of living creatures. Most of the experimental part of the work is focused on the development of tailored apatite layers on the surface of starch based biodegradable polymers, using a sodium silicate gel as an alternative nucleating agent. This methodology proved to be a very effective and simple mean of inducing the formation of a welldefined apatite-like layer at the surface of these polymers. One of the advantages when comparing with the traditional biomimetic coating methodology is that apatite is formed at a higher rate. Preliminary cell studies on these coatings have indicated a very good osteoblast adhesion and proliferation. When increasing the ionic product of the Simulated Body Fluid solution (SBF), it was possible to positively influence cell proliferation possibly due to an increase in the apatite crystallinity. Sodium silicate biomimetic methodology was also employed to coat the surface of starch based scaffolds produced by a rapid prototyping technology (Bioplotter®), recently proposed for bone Tissue Engineering applications. This coating methodology is particularly suitable for complex-shaped materials since sodium silicate gel can reach the inner surfaces of the porous structures. In this work, several dynamic biomimetic coating routes are originally proposed with the aim of producing coatings homogeneously distributed throughout the thickness of the scaffolds, namely by using static, agitation and circulating flow perfusion conditions. A bioreactor was specially designed for the last operating condition. Bone-like poorly crystalline carbonated apatite layers were effectively produced at both the external and bulk surfaces of the developed scaffolds, by covering the surface of the fibres of the scaffold while maintaining its initial porous structure and interconnectivity. The composition, chemical structure and crystallinity of the obtained apatite layers grown under static, agitation and flow perfusion conditions were not significantly different. In case of the flow perfusion, the coating thickness was greatly enhanced. Besides the typical characterization techniques, Micro- Computed Tomography analysis (µ-CT) was here used for the first time to assess scaffold porosity and as a new tool for mapping apatite content. 2D histomorphometric analysis was performed and 3D virtual models were created using specific softwares for CT reconstruction. By this technique it was possible to observe, in a non-destructive way, that the interior of the scaffolds was effectively coated without compromising their overall morphology and interconnectivity. µ-CT analysis clearly demonstrated that flow perfusion was the most effective condition. Besides mimicking better the biological milieu, it allowed for the coating of complex architectures at higher rates of apatite formation, greatly reducing the time of the coating process step. The possibility of tailoring an apatite layer by incorporating bioactive agents was also assessed in this work. A well known therapeutic agent from the family of bisphosphonates (BP) - sodium clodronate - was incorporated in an apatite coating, previously formed on the surface of a starch based polymer by a sodium silicate methodology, as a strategy to develop a site-specific drug delivery system for bone tissue regeneration. This drug, currently in use for the treatment of several bone- and calcium- related pathologies like hypercalcemia, Paget’s disease or osteoporosis, was successfully incorporated, at different doses, in the structure of a biomimetic apatite layer. It is foreseen that these coatings can for instances be produced on the surface degradable polymers for regulating the equilibrium on osteoblastic/osteoclastic activity in the direction of a regenerative effect at the interface between biomaterial and bone. The final study conducted in this work explores the possibility of incorporating Strontium into the structure of apatite coatings prepared by a solution-derived process according to an established biomimetic methodology for coating titanium based orthopaedic implants. It is believed that its localized presence at the interface between an implant and bone will enhance osteointegration and therefore, ensure the longevity of a joint prosthesis. By using this methodology it was possible to incorporate increasing amounts of Sr in the apatite layers. Sr was found to incorporate in the apatite lattice through a substitution mechanism by replacing Ca in the apatite lattice. The presence of Sr in solution induced an inhibitory effect on mineralization, leading to a decrease in the thickness of the mineral layers. The obtained Sr-substituted biomimetic coatings presented a bone-like structure similar to the one found in the human bone and are therefore expected to enhance bone formation and osteointegration. The results herein presented demonstrate the potential and versatility of the proposed biomimetic strategies for improving the performance of biomaterials either in the context of regeneration or traditional substitution of bone.