This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. There has been no change in the scope of this project. The goal remains to place/recruit bone marrow-derived cells to sites of injury. To better understand the mechanisms involved and additional ways to achieve these goals, mouse experiments have been implemented. These will provide evidence for the useful role of GCSF mobilization to help tissue repair. There are two main approaches to using bone marrow-derived stem cells to accelerate wound healing. The first approach, recently reported by our group, is to aspirate bone marrow, expand bone marrow-derived stem cell in vitro, and to physically transfer the cells to the wound site using a suitable delivery system. Using mesenchymal stem cells (MSC) delivered in a fibrin spray, we have shown accelerated healing in human chronic wounds and a possible reduction in scarring. However, this method is laborious and may require repeated bone marrow aspiration, especially for large wounds. It also leads to manipulations of the stem cells in vitro, thus requiring additional regulatory steps. We are still continuing to explore this approach, and for that purpose have established a dedicated Good Manufacturing Practices (GMP) facility. The second, possibly simpler, approach is to mobilize stem cells from the marrow using a systemically injected cytokine;however, little has been reported about the optimal conditions and parameters for this approach. We also don't know how to best direct the mobilized cells to the site of injury, although we have previously shown that fibrin and products released during fibrin polymerization are chemotactic for inflammatory cells. To answer these questions, we have begun murine studies for determining the best approaches and whether accelerated healing can be achieved by these means. The overall scope of our studies remains the same, namely to use bone marrow-derived stem cells to accelerate healing. The following are our specific aims: 1) To determine the optimal conditions for mobilizing bone marrow populations that are functionally effective for wound repair. Using different preparations of GCSF, we will study the dose and parameters that are best able to mobilize sca-1+ and c-kit+ cells, as well as CD150+/CD48- subpopulations. Flow cytometry and colony formation studies will be used to determine stem cell mobilization. Murine back wounds and dorsal tail wounds will be used to determine wound healing after mobilization. 2) To determine whether GCSF mobilization can be used to direct cells to the site of injury. We will make use of fibrin sprayed into the wound, at various concentrations. Products that are released from fibrinogen upon polymerization to fibrin with thrombin have chemotactic properties that can be used for these purposes. Immunolabeling of stem cell markers, including those for mesenchymal stem cells (CD34-/CD45-;CD29+/CD90+), will be used in wound biopsy specimens. This approach can be complemented by the use of chimeric animals. 3) To characterize and closely correlate the expression of wound edge molecular markers of impaired healing and epithelial migration in response to treatment. Baseline and sequential biopsies from the edges of murine wounds will be used to determine the epidermal expression of c-myc, b-catenin, and keratins 6/16 and 17 at the wounds'edges. These measurements will help us establish molecular markers involved in healing and whether stem cell treatment may work by affecting the expression and localization of these specific molecular markers. We conclude that there are optimal conditions to achieve the treatment of wounds with bone marrow-derived stem cells.
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