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.
BROADER IMPACT An important aspect of this research plan is the continuous integration of education and outreach programs to effectively train students, teachers, and researchers at multiple levels. This section details the project outcomes from this effort. K-12 Videoconference Education: Throughout this project, the PI has been working with middle school and high school students through Direct to Discovery (D2D), a program that she helped to establish. This program allows researchers at Georgia Tech (GT) to share cutting-edge research with students in their classrooms using high-speed internet. A number of Georgia schools have already incorporated videoconference equipment in their classrooms to connect to GT via the PeachNet, a high-speed research and education network. Many of these schools are in rural areas that would not have access to GT resources without D2D. The D2D program also received funding from Georgia’s Race to the Top program, which was used to purchase videoconference equipment and infrastructure for my laboratory and K-12 classrooms. The PI interacted with 3-5 classrooms per year through a series of D2D modules designed to be incorporated in their middle school or high school science curricula. For each module, the PI generated pre-lecture materials (worksheet with vocabulary and short reading assignment), an interactive K-12 level lecture, and experimental materials (including slides, images, videos, and/or experimental activities) for the students to analyze after each lecture. Hands-On Laboratory Training: This broader impact plan has impacted a large number of students, including underrepresented minority students, at different levels of their education through academic research and student participation in outreach activities and videoconference interactions. Four graduate students (including 1 minority student and 1 female student) and 30 undergraduate students (including 11 minority students and 17 female students) have been included in research activities. Undergraduate students were recruited from GT and minority research programs, including the NSF-funded Summer Undergraduate Research Experience (SURE) (for minority students outside of Georgia) and the Summer Transfer Enrichment Program for Science Majors (STEPS) (for minority students at local colleges) (3 SURE and 3 STEPS students in all). The PI also provided 12-week internships for 3 local high school students, including two students from Wheeler High School (including 1 minority student) and one female student from Dunwoody High School. Course Development: The PI developed an advanced undergraduate and graduate course entitled Biomolecular Engineering of the Cell. This course is a 3-unit elective that applies engineering principles to the quantitative analysis of biological systems. Chemical engineering principles of transport phenomena, thermodynamics and kinetics were integrated with cellular and molecular biology to provide strategies for engineering cells and characterizing their ability to function as therapeutic vectors. INTELLECTUAL MERIT Mesenchymal stem cells (MSCs) are multipotent stem cells that spontaneously home from the bone marrow to inflamed tissues and tumors; however, extended culture and genetic manipulation, required for therapeutic development, reduces this homing ability, limiting their use in the development of cell-based therapeuticsThis project was focused on the optimization of MSC migration in the wound bed, which is important in the development of stem cell based therapeutics for chronic wounds. In vitro techniques were used to characterize relevant mechanical and adhesive properties of MSCs and an in vivo wound healing model was used to characterize MSC migration in the wound bed. This study has resulted in more than 5 publications; results from two of these papers are summarized below. Experimental Cell Research 319: 684-696 (2013). This paper details the mechanical and functional differences in early passage and late passage human MSCs. These studies show that even before differences in classical senescence markers emerge, human MSCs undergo a loss in their ability to respond to chemical stimuli by altering their mechanical properties, which is correlated with reduced cell migration and collagen contraction. Analysis of the cytoskeleton that determines these mechanical properties revealed thickening of actin stress fibers, as well as decreased adhesion. An in vivo wound healing model was used to demonstrate that reduced mechanical responsiveness correlates with a decreased ability of MSCs to aid in wound closure. Stem Cells and Development 23(3): 245-61 (2014). PDGF and TGF-β1 are present in wounds and play an important role in recruiting MSCs and influencing their growth and regenerative capacity. This study details the mechanical and chemical responses of MSCs to these specific soluble growth factors. TGF-β1 alone, or in combination with PDGF, induced cytoskeletal remodeling to change cell shape and intracellular rheology and to increase integrin-dependent adhesion. Although this mechanical response was primarily in part to TGF-β1, combination of PDGF with TGF-β1 enhanced these changes significantly.
