Engineering Mesenchymal Stem Cells for More Rapid Wound Healing

1066585DawsonIntellectual Merit：皮肤损伤导致伤口愈合过程的激活，这是一系列复杂的事件，包括炎症、肉芽组织的产生、再上皮化和疤痕组织的形成。生长因子在伤口愈合过程中起着至关重要的作用，从表皮损伤到瘢痕组织形成这一过程是连续不断的。老年患者和慢性疾病患者，当伤口愈合过程处于慢性炎症状态时，就会形成慢性伤口。与慢性伤口形成有关的两个因素是：(1)生长因子的产生受损；(2)血管生成减少。细胞疗法，包括成纤维细胞和间充质干细胞（MSCs），以前被用于促进更快的伤口愈合；然而，这些疗法的有效性受到细胞在伤口床上迁移和植入不良的限制。我们的研究将集中在优化MSC在伤口床上的迁移。据推测，迁移的间充质干细胞在整个伤口床上扩散，有助于肉芽组织的形成，肉芽组织会收缩伤口，使伤口更快愈合。骨髓间充质干细胞迁移的改善也可能改善生长因子的时空活性，因为它们将从散布在整个伤口组织的骨髓间充质干细胞中分泌出来。血小板分泌的可溶性蛋白，如PDGF和TGF- Ò？介导骨髓源性细胞向创面组织的迁移；然而，对于这些蛋白对间充质干细胞微观力学性能的影响知之甚少。利用定量实时显微镜技术，包括颗粒跟踪微流变学和延时荧光显微镜，研究PDGF、TGF-?Ò？和/或缺氧对细胞内流变学、细胞骨架组织和与细胞粘附分子的相互作用的影响将被监测。体外跨井迁移研究将用于测量选定刺激对MSC迁移的影响。这些技术将共同用于确定迁移间充质干细胞的机械和粘附性能。优化后的MSCs，被发现具有与增加迁移相关的机械特性，将在伤口床上进行体内迁移测试。然后，msc将通过基因工程来表达血管内皮生长因子，这将用于促进更快的血管生成。提出的研究将产生关于血小板分泌的可溶性蛋白对间充质干细胞的机械和粘附性能的影响的基本信息。这些信息将用于开发基于间质干细胞的伤口愈合疗法。

1066585DawsonIntellectual Merit: Injury to the skin results in the activation of the wound healing process, a complex series of events that includes inflammation, production of granulation tissue, re-epithelialization, and scar tissue formation. Growth factors are essential in mediating the stages of wound healing, which proceed continuously from the time of epidermal damage to scar tissue formation. Elderly patients and patients suffering from chronic illness develop chronic wounds when the wound healing process is arrested in a state of chronic inflammation. Two of the factors that have been associated with the formation of chronic wounds are: (1) impaired production of growth factors and (2) reduced angiogensis. Cell-based therapies, including fibroblasts and mesenchymal stem cells (MSCs), have previously been used to promote more rapid wound healing; however, the effectiveness of these therapies was limited by poor cell migration and engraftment in the wound bed. The proposed studies will focus on the optimization of MSC migration in the wound bed. It is hypothesized that migrating MSCs that disseminate throughout the wound bed will contribute to the formation of granulation tissue, which will constrict the wound for more rapid wound closure. Improved MSC migration may also improve the spatio-temporal activity of the growth factors since they will be secreted from MSCs disseminated throughout the wound tissue. Platelet-secreted soluble proteins, such as PDGF and TGF-?Ò?¡, mediate the migration of bone marrow derived cells to wound tissues; however, little is known about the effects of these proteins on the microscopic mechanical properties of MSCs. Using quantitative real-time microscopy techniques, including particle tracking microrheology and time-lapsed fluorescent microscopy, the effects of PDGF, TGF-?Ò?¡, and/or hypoxia on intracellular rheology, cytoskeletal organization, and interaction with cell adhesion molecules will be monitored. In vitro transwell migration studies will be used to measure the effects of selected stimuli on MSC migration. Together these techniques will be used to determine the mechanical and adhesive properties of migratory MSCs. Optimized MSCs, found to possess the mechanical properties associated with increased migration, will be tested in vivo for migration in the wound bed. MSCs will then be genetically engineered to express vascular endothelial growth factor, which will be used to promote more rapid angiogenesis. The proposed studies will generate fundamental information about the effects of platelet secreted soluble proteins on the mechanical and adhesive properties of MSCs. This information will be utilized in the development of MSC-based therapies for wound healing.