The application of cell electrophoresis on the analysis of differentiation of mesenchymal stem cells induced by mechanical stress and/or co-culture system

• Main Authors: I-Chi Lee, 李亦淇
• Other Authors: Tai-Horng Young
• Format: Others
• Language: en_US
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/18907132498018010590

博士 === 國立臺灣大學 === 醫學工程學研究所 === 95 === The anterior cruciate ligament (ACL), an intraarticular ligament of the knee, is important for knee stabilization. Unfortunately, it is the most commonly injured intraarticular ligament. So far, the therapeutic options to repair torn ligaments are tissue reconstruction using autograft or allograft, reparation alone or with augmentation, or replacement using a synthetic prosthesis. Unfortunately, none of these surgical alternatives provides a long-term adequate solution. Therefore, the high incidence of ACL failures, lack of capacity for self-repair, and limitations of current treatment options have driven the research into ligament tissue engineering as a new option. Mesenchymal stem cells (MSCs) can differentiate into multiple non-hematopoietic cell lineages, including osteoblasts, chondrocytes, and ligament cells. Even in older individuals, bone marrow stroma is relatively easily harvested and contains biosynthetically active precursors and multipotent cells. This study proceed with the tissue engineering of ACL involving the isolation and identification of ACL cells and MSCs, finding the appropriate mechanical stretch to promote the up-regulation of ACL cells’ ECM gene expression of MSCs, and provide the necessary biophysical regulatory signals to induce MSCs differentiation. In Chapter 1, we introduce the development and characteristic of MSCs, besides, the progress and the problems of ACL tissue engineering are also introduced. Ligaments have a unique combination of molecular, structural, and mechanical properties, but there is no single unique marker that can be used to distinguish between ligaments and other tissues. Therefore, the development of a useful technique to discriminate ACL cells from MSCs is very important in ACL tissue engineering. In Chapter 2, the purpose of this chapter is to identify the difference between MSCs and ACL cells for the application of distinguishing theses two types of cells during the process of MSCs differentiating into ACL cells. Surprisingly, cell electrophoresis could distinguish MSCs from ACL cells successfully. Although various traditional methods such as flow cytometry, immunocytochemistry, and RT-PCR have been developed to determine the expression of specific proteins or genes to characterize different cells, they cannot be used to distinguish MSCs and ACL. In addition, using traditional methods to identify cells is not only expensive but also time consuming. Cell electrophoresis, measuring the electrophoretic mobility (EPM) of cells, is proposed to investigate the discrepancy of surface charge property of MSCs and ACL cells. Although cell electrophoresis cannot be used to determine the specific surface protein, EPM can reflect the net surface charge density of cell membrane, which can be influenced by the dissociation of functional groups of peripheral membrane proteins. It is suggested that cell electrophoresis, while simple in manipulation, can serve as a useful research tool to assist cell identification. Differentiation of MSCs into different kinds of cells is regulated by many factors, such as growth factors, cytokines, and hormones. Mechanical stretch has been shown to affect the healing and remodeling process of ligament, bone, and cartilage. Many articles have been published which discussed the effects of mechanical stretch on various cell types, however, little is known about the effects of mechanical stretch on differentiation of MSCs. In Chapter 3, in order to determine the correlation of mechanical stimulation and differentiation tendency in vitro, three groups of cell markers, bone, tendon/ligament and cartilage, are elongated on 3% and 10% using the Flexcell stress system, without addition of other growth and differentiation factors. The results reveal that mechanical stretch couldn’t induce MSCs into cartilage cell lineage. In contrast, the bone cell lineages markers are up-regulated at strains of low magnitudes and the tendon/ligament cell lineages markers are up-regulated at high magnitudes strain. Therefore, this study shows that mechanical stretch is an effective factor that could regulate the early stage differentiation pathway of MSCs into bone or ligament/tendon cell lineages. Furthermore, since most of the mechanisms of induction MSCs to desire cells are clearly understood, the induction mechanism of MSCs to ACL cells is still not obvious. Differentiation of MSCs into ACL cells is regulated by many factors. Since mechanical stress affects the healing and remodeling process of ACL after surgery significantly, co-culture system had also showed the promise to differentiate MSCs toward different kinds of cells on current research. In Chapter 4, we investigate the gene expression of major extracellular matrix component molecules of ACL cells, collagen type I, type III, and tenascin-C of MSCs under three induction groups. In addition, to follow the study on chapter 2, cell electrophoresis technique and mRNA level gene expression of MSC protein are also used to analyze the differentiation of MSCs. Group I is the MSCs co-culture with ACL cells. Group II is the MSCs exposure to mechanical stress. Group III is the MSCs exposure to mechanical stress after co-culture with ACL cells. The results reveal that specific regulatory signals releasing from ACL cells appear to be responsible for supporting the selective differentiation toward ligament cells in co-culture systems and mechanical stress promotes the secretion of key ligament ECM components. In addition, the results also reveal that combined regulation of mechanical stress and co-culture effect could assist the development of healing and remolding of ACL tissue engineering. Furthermore, this study also demonstrates that cell electrophoresis could be used in investigation of cell differentiation. Also, analysis of the data suggests the feasibility of utilizing MSCs in clinical applications for repairment or regeneration of ACL tissue.