Generation of mineralised cellular constructs using mouse embryonic stem cells encapsulated in alginate hydrogels and cultured within a custom-made rotating wall vessel perfusion bioreactor

摘要

The prevalence of musculoskeletal disorders is a major burden for modern societies. Due to the increasing aging population and the lifestyle changes, a significant number of people are severely affected worldwide. The important issue with these diseases is the fact that they cause pain and disability on a person's physical functioning for long time, thus diminishing the quality of life of the individual. Moreover, they are accompanied by a high financial cost for the society and the healthcare system. Current ways of treatment do not provide optimum therapy. They employ high concentration of growth factors and they are expensive, inefficient and many times exhibit various side effects. For this reason, an alternative solution is needed. udTissue engineering (TE) strategies offer a novel approach to the problem. The combination of the appropriate cell source with the essential scaffold leads to the formation of three-dimensional (3D) constructs, which can be subsequently, cultured within a bioreactor, with the employment of proper osteoinductive factors. This process leads to the generation of high number of efficiently differentiated cells, which are needed for cellular therapies. udIn this project, the generation of 3D mineralised cellular constructs was performed using mouse Embryonic Stem Cells (mESCs) encapsulated in alginate hydrogels. The novelty of this project lied on two components; the employment of a custom-made rotating wall vessel (RWV) perfusion bioreactor, which had been shown previously to exhibit advantageous properties regarding the efficient differentiation of high cell numbers needed for potential therapeutic applications and the use of simvastatin (Sim) in the culture media, acting as an osteoinductive substance in very low concentration of the nanomolar scale. Sim had been previously employed to induce osteoblast differentiation. The novelty lied on the total combined configuration with the low concentration of Sim and the perfusion bioreactor used for cell culture and differentiation. udEvaluation of cell proliferation and osteogenic differentiation was performed through several analyses. Extended gene expression was tested and obtained results were also compared with those acquired previously by the currently used protocol with dexamethasone (Dex). Acquired results indicated that the favorable environment of the perfusion bioreactor culture could support higher cell number sand more efficient osteogenic differentiation in comparison to static configuration. Sim was more efficient when supplied in the culture at the appropriate time point, after two weeks of initiation of the experiment. Sim and Dex indicated similar outcome in biochemical analysis. Osteogenic gene expression was strongly induced after Dex treatment while Sim supported the generation of higher cell numbers. These findings suggested the generation of a more progenitor cell type after Sim treatment and a more mature phenotype after Dex treatment.