Association of tissue engineering techniques for mineralized tissue regeneration under degenerative inflammatory stimulus: analysis on 3D-culture perfusion bioreactor and animal inflammatory models

Abstract

The regeneration of mineralized tissues under degenerative inflammatory stimulus is a challenging situation, since the reparative potential of resident stem cells may be negatively influenced in the presence of high dosage of pro-inflammatory mediators. In this research project, an association of tissue engineering techniques will be evaluated in order to develop macro-porous chitosan scaffolds with architecture and composition similar with those found on mineralized tissue extracellular matrix, associated with release of pleiotropic drugs (statins) in order to modulate inflammation, angiogenesis and neo-tissue genesis by resident cells. Tissue engineering techniques will also be applied for the development of a realistic 3D-cell culture model, to evaluate the scaffolds at specific application sites in vitro. Initially, an in vitro degenerative inflammatory model capable of reduce in around 50% of mineralized tissue deposition by bone marrow (BMSC) and dental pulp (DPSC) stem cells will be stablished, by means of co-culture with LPS-stimulated monocytes (Phase 1). Then, the anti-inflammatory, pro-angiogenic and bioactive dosage of statins (simvastatin, atorvastatin and rouvastatin) will be selected. The effect of these dosage associations on the regenerative potential of BMSCs and DPSCs under degenerative inflammatory stimulus will be assessed (Phase 2). After that, several parameters will be tested to develop a macro-porous nano-fibrous chitosan scaffold with an interconnected pore network. This scaffold will be biomineralized with hidroxiapatite, in order to simulate mineralized matrix chemical composition. A drug release system based on chitosan microspheres loaded with the anti-inflammatory and bioactive dosage of statin will be added to the scaffold (Phase 3). The biologic effect of the scaffold will be assessed on BMSCs and DPSCs under the inflammatory stimulus, as well as the pro-angiogenic potential with endothelial cells (Phase 4). Finally, two in vitro cell culture models will be developed to evaluate the regenerative potential of the scaffolds at specific application sites (Phase 5). Bovine cancellous bone and dental pulp tissue samples will be collected and subjected to the decellularization process followed by BMSC or DPSC seeding, in co-culture or not with endothelial cells, on perfusion bioreactor. The scaffolds will be positioned on specific sites created on the tissue constructs, and this set will be cultivated on the perfusion bioreactor system. Finally, the effect of the scaffolds at specific application sites on animals will also be assessed (Rattus novergiccus) by means of experimental models of chronic inflammation. (AU)