Fat Grafting Supplemented With Platelet Rich Plasma Overcomes Oral Fibrosis In A Prrx1 Mouse Model
Michelle Griffin, MD PHD1,2, Nicholas Guardino, BS1, Amanda Spielman, BS1, Darren Abbas, MD1, Derrick Wan, MD1, Michael Longaker, MD1.
1Stanford University, Stanford, CA, USA, 2University College London, London, United Kingdom.
PURPOSE:Oral scarring is common side effect of head and neck radiation therapy causing long term pain and taste disturbance for patients. Fat grafting has shown to improve the outcomes of irradiated skin. Platelet rich plasma (PRP), consists of growth factors which have shown to improve skin fibrosis. We have previously identified Prrx1 fibroblasts as having a role in fibrosis in mice. Using a radiation oral murine model, we aimed to understand the regenerative effects of fat grafting supplemented with PRP in oral irradiation.
METHODS:Adult (60-day old) Prrx1Cre;R26mTmG transgenic mice (n=24) underwent total body irradiation with 9 Gy for immunodepletion. Mice were then immediately reconstituted with 2 million nucleated bone marrow cells from donor NSG(NOD.CB17-Prkdcsscid/J) mice via retro-orbital injections. Reconstitution was confirmed with fluorescence-activated cell sorting (FACS) 2 weeks after whole body irradiation. The mice were then randomized to one of four conditions (n=6/per group): 1. Oral cavity irradiated with 5 Gy every other day for 12 days (30 Gy total) (Control group), 2 Irradiation to the oral mucosa with simultaneous treatment with human lipoaspirate (Fat group), 3. Irradiation to the oral mucosa with simultaneous treatment with human lipoaspirate + PRP (Fat + PRP group), and 4. Irradiation to the oral mucosa with simultaneous treatment with PRP (PRP group). Mice were then euthanized at 4 weeks post irradiation for histological and flow cytometry analysis (FACS). Oral sections underwent immunofluorescent staining for fibrosis (e.g., collagen type I/col-I, alpha smooth muscle actin/a-SMA), vascularization (CD31 staining), and inflammation (F4/80 antigen) and were imaged on a confocal microscope.
RESULTS:Two weeks post bone-marrow transplant, >90% of circulating hematopoietic cells (CD45+) cells in prrx1Cre;R26mTmG reporter mice were non-fluorescent, signifying successful reconstitution. At 4 weeks post irradiation Prrx1+GFP cells were significantly reduced by FACS in the Fat + PRP group than irradiated mucosa treated with PRP or Fat grafting alone (p < 0.05), signifying less presence of Prx1+ fibroblasts. Histological confirmation revealed reduced dermal thickening and collagen deposition in the mucosa treated with Fat + PRP compared to PRP or Fat grating alone (p < 0.05). Lastly immunohistochemistry demonstrated improved vascularisation with greater CD31 staining and reduced inflammation with decreased F4/80 staining in the Fat + PRP group compared to the Control group (p < 0.05).
CONCLUSION:Our novel murine model suggests that fat grafting is beneficial in reducing the burden of oral fibrosis. A combination of PRP and fat holds promise to improving the outcomes of radiation induced fibrosis by reducing pro-fibrotic fibroblasts.
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