Endothelial Differentiation Of Bone Mesenchymal Stem Cells And The Effects Of Scaffold On It

Cardiovascular diseases are the leading cause of mortality worldwide, it often requires surgical intervention with bypass. For small-diameter (<6mm) blood vessel, vascular scaffolds lack a long-term patency rate due to the liable possibility of intimal hyperplasia and thrombosis. Surface endothelialisation can provide an effective alternative to improve the function of vascular scaffolds. However, endothelial cells (ECs) have some shortages such as the source limitation and proliferative limitation of terminally differentiated cells. In contrast, bone mesenchymal stem cells (BMSCs) shed new insights into vascular cell regeneration based on its inherent ability of self regeneration, reliable isolation method and differentiation potential to ECs, showing promise in overcoming these limitation. Besides, the surface modification of synthetic vascular scaffolds by using biomimetic peptides has also shown a promising strategy to improve the scaffold endothelialisation.In our previous studies, a three-dimensional nanofiber scaffold consisting of RGD spider silk protein (pNSR32), polycaprolacton (PCL) and chitosan (CS) was prepared by electrospinning. The pNSR32/PCL/CS small diameter vascular scaffold showed a good biocompatibility and mechanical properties. When applied in artificial coloboma surgery in abdominal aortic of SD rats, the scaffold kept intact and was of favorable hemokinesis ability at8w in vivo. Hence, the pNSR32/PCL/CS scaffold can function as a promising scaffold source for vascular tissue engineering.The aims of this study were to investigate the in vitro differentiation of SD rats BMSCs (rBMSCs) to ECs and the effects of pNSR32/PCL/CS scaffold on endothelial differentiation of rBMSCs and biological function of induced endothelial cells (IECs). The specific contents shown in this thesis are followed as:1. rBMSCs was isolated based on the property of direct plastic adherence. The cultured cells showed a typical characteristic of rBMSCs with fibroblastic-shape, and with the CD90+CD105+CD45"CD34-CD11b-characteristics and surprising multilineage potential. Moreover, the cytoskeletal protein of rBMSCs displayed long, parallel. In addition, the S-shaped proliferation curves and27.71%proliferation index in analysis of cell cycle indicated the strong proliferation activity of rBMSCs. 2. By the promotion of inductors VEGF and bFGF, rBMSCs were differentiated to IECs directionally, which was verified by the evidence from which endothelial-specific marker vWF expression was found, together with the ability of formation of capillary-like structures and NO release.3. After3weeks endothelial induction, the relative Pecaml expression of IECs cultured on PCL/CS showed a signifiant decrease compared to that on pNSR32/PCL/CS, which was similar to ECs on TCP. Several identifying tests including cell adhesion rate and cytoskeletal protein staining revealed that pNSR32/PCL/CS and PCL/CS were of good matrixes for IECs attachment and spreading, while IECs attached on pNSR32/PCL/CS were more stretched, connecting to neighboring cells. When compared with PCL/CS and TCP, the pNSR32/PCL/CS scaffold significantly increased NO secretion by IECs. IECs cultured on pNSR32/PCL/CS showed a2-fold increase in Angl expression, which played essential role in both angiogenesis and maturation.