可注射式細胞載體於心肌組織工程的應用

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可注射式細胞載體於心肌組織工程的應用

Injectable Cell Delivery Vehicles for Myocardial Tissue Engineering

葉怡君 , Ph.D　　Advisor：宋信文　　

英文

細胞治療；羊水幹細胞；細胞片工程；可注射式載體

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Myocardial infarction (MI) progresses from the acute death of cardiomyocytes and the infiltration of inflammatory cells into granulation, followed by scars. Recently, cell transplantation via local intramuscular injection is a promising therapy for patients with MI. However, the time required to process and expand autologous cells in vitro makes applying these cells in patients with an acute MI in a timely fashion difficult. Moreover, following intramuscular injection, retention of the dissociated cells in the transplanted area remains problematic. These facts can be deleterious to cell transplantation therapy.
The purpose of study I to evaluate using human amniotic fluid stem cells (hAFSCs) derived from second-trimester amniocentesis for the therapeutic potential of cardiac repair. Whether hAFSCs can be differentiated into cardiomyogenic cells and toward the maturation of endothelial cell lineage was investigated in vitro using mimicking differentiation milieu. Test samples were xenogenically transplanted into the peri-ischemia area of an immune-suppressed rat model at 1 week after MI induction. There were three treatment groups (n ≥ 10): sham; saline; dissociated hAFSCs. After 4 weeks, hAFSCs-treated animals showed a preservation of the infracted thickness, an attenuation of left ventricle remodeling, a higher vascular density, and thus an improvement in cardiac function. 
Study II examined the hypothesis that the thermoreversible methylcellulose hydrogel was used as a coating on TCPS dishes and developed for harvesting living hAFSCs sheets. The optioned hAFSCs sheets preserved the intercellular junctions and endogenous extracellular matrix (ECM) and retained the cell phenotype. Test samples were xenogenically transplanted into the peri-ischemia area of an immune-suppressed rat model at 1 week after MI induction. There were four treatment groups (n ≥ 10): sham; saline; dissociated hAFSCs; and hAFSCs sheet fragments. The results obtained in the echocardiography revealed that the group treated with hAFSCs-sheet-fragment had a superior heart function to those treated with saline and dissociated hAFSCs group (P < 0.05). Due to their inherent ECM, hAFSCs sheet fragments had a better ability of cell retention and proliferation than dissociated hAFSCs upon transplantation to the host myocardium. Additionally, transplantation of the hAFSCs sheet fragments stimulated a signification increase in vascular density, consequently contributing towards improved wall thickness and a reduction in the infarct size. Our histological findings and qPCR analyses suggest that the transplanted hAFSCs can be differentiated into cardiomyocyte-like cells and cell of endothelial lineaged and modulate expression of multiple angiogenic cytokines and cardiac protective factor with the potential to promote neo-vascularization, which evidently contributed to the improvement of ventricular function. These results suggest that the use of fragments of cell sheets can serve as a cell-delivery vehicle by providing an adequate physical size and a favorable ECM environment to retain the transplanted hAFSCs in the infracted myocardium. The benefit effects of hAFSCs may be mediated not only through their ability to differentiate into cardiomyocyte-like cells and cells of endothelial lineages but also through the growth factor-mediated paracrine regulation.
In our previous study, the number of cells on the 50-mesh-sized for 27 gauge optimize injection condition is 65 cells/fragment. The ideal number of cell transplantation for clinical use is around from 1×106 to 1×107. Recently, the use of three dimensional multicellular aggregates (cell bodies) for myocardium infarction was investigated. For the human amniotic fluid stem cells, 1×104 cells on the methylcellulose hydrogel can form the cell body and the diameter can reach to 350 μm. However, it has been often reported that cell necrosis occurs due to the insufficient supply of oxygen/nutrients and the accumulation of metabolic wastes, especially at the center of the cell bodies. In order to address this problem, the injectable of three dimensional porous micro-scaffolds were developed to enhance the transportation of oxygen/nutrients and wastes. In the study III, uniform porous poly(D, L-lactide-co-glycolide) (PLGA) beads with controlled pore diameters were prepared based on an emulsion templating method in a simple fluidic device. Unstable water-in-oil (W-O) emulsion, being in the course of phase-separation, was introduced into the fluidic device to form water-in-oil-in-water (W-O-W) droplets. The resultant W-O-W droplets evolved into porous PLGA beads with controlled pore diameters at the inside and surface of the beads after the organic solvent had evaporated. Porous beads prepared using the upper part of the phase-separated emulsion exhibited the large inner and surface pores as well as high interconnectivity compared to the lower part of the emulsions. Fluorescence microscopy imaging and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay confirmed the more uniform distribution and higher viability of the cells in the bead with large pores. These results suggest that the bead with large pores could provide favorable environment for cells and thus be potentially used for both tissue engineering and cell delivery system.

Reference ( 92 ) 〈TOP〉

[1] Ryu JH, Kim IK, Cho SW, Cho MC, Hwang KK, Piao H, et al. Implantation of bone marrow mononuclear cells using injectable fibrin matrix enhances neovascularization in infarcted myocardium. Biomaterials 2005; 26:319-26
連結：
[2] Perin EC, Dohmann HF, Borojevic R, Silva SA, Sousa AL, Mesquita CT, et al. Transendocardial, autologous bone marrow cell transplantation for severe, chronic ischemic heart failure. Circulation 2003;107:2294-302.
連結：
[3] Kim WG, Park JJ, Chung DH, and Na CY. Autologous cardiomyocyte transplantation in an ovine myocardial infarction model. Int J Artif Organs 2002;25:61-6.
連結：
[4] Smits AM, van Laake LW, den Ouden K, Schreurs C, Szuhai K, van Echteld CJ, et al. Human cardiomyocyte progenitor cell transplantation preserves long-term function of the infarcted mouse myocardium. Cardiovasc Res 2009;83:527-35.
連結：
[5] Yoo, KJ, Kim, HO, Kwak, YL, Kang, SM, Jang YS, Lim SH, et al. Autologous bone marrow cell transplantation combined with off-pump coronary artery bypass grafting in patients with ischemic cardiomyopathy. Can J Surg 2008;51:269-75.
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