Development of osteoconductive coatings for non-metallic bone implants
The design of osseous implants, either load bearing or not, with desired mechanical and surface features that promote integration with bone and avoid risks of bone resorption and implant failure due to shear stresses, is still a challenging endeavour. The mechanical stresses which the skeleton undergoes affect bone formation and resorption processes. Bone remodelling is often promoted by adequate stress/strain conditions which are able to prevent bone mass loss. The largely used metallic implants offer several advantages like easy shape casting and modelling but include also several drawbacks like high stiffness if compared with the mechanical properties of native bone. A new generation of bone prosthesis is therefore indispensable to overcome the limitations of the obsolete metallic devices. In the orthopaedic framework, promising results have been achieved in the recent decades by three-dimensional structures named scaffolds. It is mandatory for any optimal scaffold to act as a temporary three-dimensional support for cell adhesion, growth and mineral matrix deposition. Moreover, ideal scaffolds should be able to integrate into surrounding tissue and mimic the structure and morphology of the natural bone tissue. Strict requirements for scaffolds are biocompatibility, a design closely resembling the natural extracellular matrix, an appropriate surface chemistry to promote cellular attachment, differentiation and proliferation and a sufficient mechanical strength to withstand in vivo stresses and physiological loading. Finally, the degradation of the ideal scaffold should proceed in a controlled way, keeping a sufficient structural integrity until the newly grown tissue has replaced the scaffold's supporting functions. Coupling a three-dimensional porous scaffold and a load bearing structure with suitable mechanical properties it is possible to obtain a device where the osteoconductive and osteoinductive properties of the former are synergistically linked with the mechanical ones of the latter. In this work both the aspects – osteointegration and load bearing – of an ideal prosthesis have been investigated. Alginate/Hydroxyapatite composite scaffolds were developed to be used either as scaffolds for sub-critical defects or as coatings for load bearing non-metallic bone prostheses. In both cases the investigation aimed to select suitable components and casting procedures to obtain the best results. The features of the single components and of the final three-dimensional structure were extensively investigated in order to obtain the most clarifying characterization both in terms of physical-chemical properties and in terms of biological responsiveness. The experimental section of this work involved physical-chemical analysis that helped to characterize both the organic and the inorganic components of the scaffold, respectively alginate and hydroxyapatite, before and after composite assembling. This investigation, based on several techniques (NMR, Rheology, XRD, Raman and TEM) allowed to characterize in detail the scaffold’s components and revealed the possibility of using the hydroxyapatite as a source of calcium ions for the gelification of the alginate without loosing the paramount osteoinductive properties of the mineral. Micro Computed Tomography (µ-CT) was employed to understand quantitatively the architectural features of the three-dimensional matrix obtained after alginate gel casting process. Moreover, this tool allowed to assess the influence of different manufacturing protocols (e.g. concentration of the components, casting temperatures) on the scaffold’s final structure. The results obtained by means of µ-CT coupled with the ones of Scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) analysis of the scaffolds showed an optimal interconnected porous structure with pore sizes ranging between 100 m and 300 m and over 88% porosity. Proliferation assays and SEM observations demonstrated that human osteosarcoma cell lines were able to proliferate, maintain osteoblast-like phenotype and massively colonize the scaffold structure. Once the in vitro behaviour of the structure was clear, in vivo tests were performed. Cone-like Alg/HAp scaffolds were tested on skeletally mature female New Zealand White rabbits and compared with positive (bioactive glass scaffold) and negative (without any implant) controls. Ex vivo investigations of the dissected samples were based on µ-CT and histological analysis and revealed high level of osteointegration and osteoconduction of the scaffolds. Moreover, efforts have been made to link the porous structure to the non-metallic fibre reinforced composite used as load bearing unit. Overall, these combined results indicate that the structure here developed is promising for being employed in orthopaedic applications.