|2009 Annual Meeting Abstracts
Development of a Porcine Delayed Wound Healing Model and its Use in Testing a Novel Cell-Based Therapy
Ivan Hadad, M.D.1, Brian H. Johnstone, Ph.D.2, Jeffrey G. Brabham, M.D.3, Matthew W. Blanton, M.D.1, Pamela I. Rogers, RLATg2, Cory J. Fellers2, James L. Solomon, B.A.2, Steffi Merfeld-Clauss, B.A.2, Colleen M. DesRosiers, Ph.D.3, Joseph R. Dynlacht, M.D.3, John J. Coleman, M.D.4, Keith L. March, M.D., Ph.D.1.
1Indiana University, Department of Surgery, Indianapolis, IN, USA, 2Indiana Center for Vascular Biology and Medicine, Indianapolis, IN, USA, 3Indiana University, Department of Radiation Oncology, Indianapolis, IN, USA, 4Indiana University, Division of Plastic Surgery, Indianapolis, IN, USA.
PURPOSE: Some of the most difficult wounds to manage are those occurring in previously irradiated fields, where the frequency of complications can be as high as 67% and the patients have few options for effective treatment. Thus, there is a need for novel therapeutic agents that facilitate wound healing in similarly compromised regions. The aims of the present study were to create a delayed healing, full-thickness wound model and then use this model to test the potential for new therapies using autologous stem cells and blood-derived products.
METHODS: Using the equation described by Barton et al, alpha-beta calculations were performed to approximate the single-fraction equivalent dose of 54 - 70 Gy of external beam radiation therapy admininstered in the 1.8 Gy fractions used clinically. Single-fraction doses of 16, 18, and 20 Gy were ultimately chosen. The paraspinal skin of three female Yorkshire cross domestic swine was treated with either 16, 18 and 20 Gy in a single dose using 6 MeV electrons. Immunohistopathological analysis indicated that the 20 Gy dose has the most profound effect, with a 75% decrease in microvessel density within the dermis compared to normal, non-irradiated skin (P<0.05), and this effect stabilized between 7 and 10 weeks. There were no local or systemic complications. In the main study, 4 pigs received a single 20 Gy fraction of 6 MeV electrons to the dorsal skin surface. Five weeks after radiation, dorsal hump fat was harvested from each animal and processed to isolate autologous adipose stromal cells (ASCs), which were cultured for 2 weeks. Two weeks after fat harvest, approximately 28 full-thickness, 1.5 cm2 wounds were made in the irradiated skin fields and in non-irradiated skin of each pig. Wounds were randomly assigned to five groups: saline, ASCs in saline, platelet-rich plasma (PRP) fibrin gel, ASCs in PRP, and non-autologous GFP-labeled ASCs in PRP. On post-wounding days 4, 8, 12, 16, and 21, wound size was assessed.
RESULTS: There was a significant difference in the rate of healing between irradiated and non-irradiated skin treated with saline (p<0.05), corresponding to an 11.2% (approximately 10 day delay) in wound closure compared to wounded non-irradiated tissues. The ASC in PRP treated wounds enhanced the wound healing rate by 10% (2.5 days), compared to saline treatment (p<0.05). An enhanced healing rate was dependent on embedding the ASCs in PRP, since no improvement in healing rate was observed with ASCs suspended in saline. GFP labeling demonstrated that the ASCs persisted in the wounds.
CONCLUSION: We have created model that closely simulates a clinically relevant delayed healing environment. This model could be used to evaluate the efficacy numerous topical wound healing products. We demonstrate that a combination of ASCs and PRP cooperatively improve the healing rates of perfusion-depleted tissues, possibly through enhancing local levels of growth factors.