The effect of acellular matrix-coated nanofibrous scaffolds on differentiation of mesenchymal stem cells to neuronal cells
Project no. 21755 · Status: Kapanmış (Yayın bekleniyor) · 2020
Summary
<p>Damage to the central nervous system (CNS) is an important problem for both the individual and the society due to the limited treatment possibilities. Traumatic spinal cord injury (SCI) usually occurs due to traffic accidents, falls, and gunshot wounds. Although surgical and pharmacological methods have been tried, there is no effective treatment yet. In recent years, combinatorial approaches including cells, biomaterials and decellularized matrices have come to the fore. When the pathophysiology of SCI is examined, it is known that big changes occur in the extracellular matrix (ECM) of the injury site and an inhibitory environment for cells is formed. For this reason, it was thought that a tissue scaffold that could change the ECM of the damaged area should be used. In this thesis, we aimed to produce neuroinductive nanofibrous tissue scaffolds coated with acellular matrix.</p><p>Polycaprolactone (PCL) scaffolds (membrane, random, align) in three different surface morphologies were produced. They were coated with acellular matrix obtained from rat CNS. Fiber diameters and orientation were investigated by SEM analysis. The success of the coating was evaluated by SEM-EDS analysis, and the success of cross-linking by FTIR analysis. Neural differentiation study was carried out by seeding rat bone marrow mesenchymal stem cells (BMSCs) on the scaffolds obtained. Cytotoxicity was demonstrated by MTT analysis, neural differentiation by ßIII tubulin and GFAP immunohistochemical staining.</p><p>Fiber diameter measurements were made with SEM analysis data and dispersed fibers were found as 300-600 nm and parallel fibers 200-600 nm in diameter. More than 70% of the parallel fibers were arranged at an angle of 0±10°. In the characterization tests performed after decellularization, it was shown that the cells and their remnants were removed, the total protein amount was preserved in the brain and spinal cord, 13% and 24% respectively. It was found that 19% of fibronectin (FN) was preserved in the brain and spinal cord, brain-derived neurotrophic factor (BDNF) were preserved in the brain and spinal cord, 29% and 18.5% respectively. SEM-EDS analysis data showed that the coating was successful. FTIR analysis showed successful cross-linking of hESM with genipin. The biodegradability test showed that the scaffolds obtained were degradable for >2 months. In the cytotoxicity study, the acellular matrix was found to be biocompatible with BMSCs. When PCL scaffolds were compared with each other in 3-day MTT analysis, the highest cell viability was found on dECM (decellularized ECM)-coated random fibers. The presence of hESM in fiber scaffolds facilitated cell attachment and proliferation, but had no effect on membrane scaffolds. In the neural differentiation study, ßIII tubulin expression was observed even in the control group, which did not contain any neural differentiation agent on PCL parallel fibers. Cells tended to differentiate into neuronal lineage on PCL fiber scaffolds, whereas there was glial differentiation on hESM-coated scaffolds. </p><p>The obtained data showed that a neuroinductive scaffold has been developed. Acellular matrix coating promoted attachment and differentiation of BMSCs. The obtained tissue scaffold can be used in neural tissue engineering studies.</p>