Experimental Study Of Applying Bone Marrow Mesenchymal Stem Cells As Tissue Engineering Valve Seeded Cells

At the present time, in the fields of studying artificial heart valve, the best choice is the tissue engineering heart valve (TEHV) studying. Current options of surgical heart valve replacement comprising of mechanical or biological prostheses substantially are the preferred replacement device in our clinical. Unfortunately, there are limitations as to the long term benefits of clinically available valve prostheses. Mechanical valves are associated with a significant risk of thromboembolism, and biological valves suffer from structural dysfunction because of calcification and progressive tissue deterioration. Contemporary clinically available valve prostheses basically represent nonviable structures and lack the potential to grow, to repair, or to remodel. Of particular importance to the pediatric population, homografts and all other currently available conduits and valves have no ability to grow. Tissue engineering is an evolving science joining engineering and biology in an attempt to develop replacement tissue, and represents a novel scientific concept to overcome these limitations aiming at in vitro fabrication of living heart valves with a thromboresistant surface and a viable interstitiumwith repair and remodeling capabilities.Up to now, most approaches were based on the utilization of vascular derived cells associated with certain shortcomings. Cell harvesting before seeding necessitated the sacrifice of intact vascular structures of the donor organism. Apart from that, vascular derived cells demonstrated different characteristics compared with natural valvular interstitial cells.The qualities might be vital to the development and long term function of TEHV. We have known the feasibility of isolating and expanding bone marrow mesenchymal cells (BMSCs) in vitro. More recently, it has become evident that mesenchymal stem cells can be isolated consistently from bone marrow, expanded in vitro, and differentiated to various cell phenotypes including osteocytes, chondrocytes, adipocytes, and myocytes. In search for alternative cell sources, especially with regard to future routine clinical realization of the tissue engineering concept, we identified canine BMSC as a promising candidate. The usage of BMSC may offer several advantages in 1) easy collection by a simple bone marrow puncture avoiding the sacrifice of intact vascular structures, 2) showing the potential to differentiate into multiple cell lineages, and 3) demonstrating unique immunological characteristics allowing persistence in allogenic settings. In the present study we primarily investigate the feasibility of creating functional tissue engineered heart valves on the basis of canine BMSCs and implant TEHV into the canine abdominal aorta and observe the influence in vivo environment on tissue formation and cell differentiation.Part I: BMSCs were isolated and purified from the canines bonemarrow by Percoll density gradient centrifugation and by adhering to the culture plastic. After successive culture and amplification, the growth curve was drawn. The morphology was observed under phase contrast microscope, and the some antigens were examined by immunohistochemistry stain. Results: SH2, Vimentin, a-SMA and collagen I, III,all were positive expression in naturally differentiated BMSCs, the positive percentages were (95. 10±1. 70) %, (64.35 ± 2.17)%, (78. 74 ± 0. 93)%, (78.27±1.38) %and (66.78 ±0.84)% respectively. CD34, VIII antigen and laminin were negative reaction. The induced and differentiated cells showed positive staining of laminin (50. 23 ± 2. 45) %. The positive expression of a-SMA decreased to (42. 34 ± 1. 56) %and that of vimentin increased to(86. 24 ± 2. 41) %. The phenotypes and biological characteristics of naturally differentiated myofibroblast with canine BMSCs show it was more active than that of fibroblasts and more suitable to be used as seed-cells in constructing TEHV.Part II: Optimal precondition of the scaffolds and intervals for seeding cells were investigated in vitro. The porcine acellular aortic valves obtained through a cellular extraction procedure were seeded with BMSCs and grown in vitro in a static condition. The functions of seeding cells' synthesis and secretion were evaluated. Results: We find an efficient approach of seeding cells to scaffolds. Preconditioning of scaffolds with poly-L-lysine and repeated seeding of cells by 36 hours interval can improve the efficacy of cell seeding. The BMSCs' biocompatibility of...