Abstract

Tissue engineering is an interdisciplinary field aimed to design and engineer an efficient system for tissue and organ regeneration, for instance, for bone healing, based on the combined use of scaffolds, cells, bioactive or signalling molecules. An optimal tissue engineering procedure requires materials and scaffolds fulfilling several requirements, one of those being the ability to trigger and control the crosstalk with the biological environment both in vitro and in vivo, and to induce and control the extracellular matrix production and assembling. Diatomite is one of the most abundant natural sources of hydrated amorphous silica resulting from the accumulation of diatom skeletons. Diatoms possess particular features in structure, morphology as well as composition. Interestingly, it has been recognized that the formation process of diatom skeleton is possibly related to that of human bone. In this study, we wanted to utilize diatoms as silicon donor additives in scaffolds for bone tissue engineering, having been demonstrated the important role of silicon in bone formation. In this first part of the project, we used several methods to eliminate impurities in the raw diatomite. Diatom microparticles (DMPs) and nanoparticles (DNPs) were successfully produced by fragmentation of purified diatoms under alkaline condition. Our result showed that both DMPs and DNPs were able to release silicon, as detected in-vitro by inductively coupled plasma optical emission spectrometry (ICP/OES). In addition, diatom microparticles and nanoparticles - derived from diatom skeletons - showed minimal or non-cytotoxic effects in-vitro as determined by lactate dehydrogenase assays on cell cultures. These findings suggest that diatom particles derived from diatom skeleton as a silicon donor might have potential use for bone tissue engineering. In the second part of this thesis, we studied the effect of diatom particles on some properties of silk fibroin/diatom particles scaffolds. To handle this task, a series of fibroin scaffolds loaded with different amounts and size of diatom particles (microparticles, nanoparticles and their combination) were fabricated by using the salt leaching method. Diatom particles addition influenced scaffold morphology and mechanical properties, and its biological behaviour as assessed on human osteosarcoma cell line MG63 cultures. Scaffolds loaded with diatom particles strongly enhanced cell adhesion, metabolic activity and proliferation. Moreover, the possible beneficial effect of the addition of diatoms particles to silk fibroin on early bone formation was determined through collagen type I synthesis evaluation, osterix expression and alkaline phosphatase induction. Cultures with human mesenchymal stem cells (hMSCs) demonstrated the silk/diatom particles scaffolds were able to induce the differentiation of progenitor cells. In conclusion, our findings provided strong evidence for a potential use of diatom particles- derived from natural diatom skeleton in biological applications, in particular for bone tissue regeneration.