Fat Grafting Mitigates Radiation-induced Scalp Fibrosis And Decreases The Abundance Of Profibrotic Engrailed1-positive Fibroblasts In The Overlying Skin
Mimi R. Borrelli, MBBS, MS., Sandeep Adem, MS, Nestor M. Deleon Diaz, Dung Nguyen, MD, Arash Momeni, MD, PhD, Michael T. Longaker, MD, MA, Derrick C. Wan, MD.
Stanford, menlo park, CA, USA.
PURPOSE: Radiotherapy (RT) is used to treat thousands of patients every year, however it can result in significant damage to collateral healthy soft tissue within the radiation field. Radiation-induced soft tissue fibrosis may be severe and debilitating, but despite the frequency of this complication, there are few effective treatment strategies. Fat grafting is emerging as a popular technique able to address volume deficiencies and regenerate fibrotic scar tissue. The mechanisms of improved tissue quality after fat transfer remains poorly understood. We recently identified the Engrailed-1(En1)-positive fibroblast lineage in dorsal skin and scalp of mice responsible for the majority of collagen production in acute and chronic fibrosis. Here we asked whether grafted fat could alter the composition of this profibrotic fibroblast subtype in the irradiated scalp skin of recipient mice. METHODS: Adult (60-day old) En1Cre;R26mTmG transgenic reporter mice (n=9) underwent full body lethal irradiation with 9 Gy for hematopoietic depletion. Mice were then immediately transplanted with 2 million nucleated bone-derived cells from donor NSG (NOD.CB17-Prkdcsscid/J) mice via retro-orbital sinus injection (Fig. 1A). Fluorescence-activated cell sorting (FACS) analysis of peripheral blood was used to assess the success of reconstitution. After 4 weeks, the calvarial region was then irradiated with a total of 30 Gy, fractionated into six 5 Gy doses delivered on alternative days across 12 days. A 4-week recovery period followed to allow for chronic radiation-induced skin fibrosis to develop. The irradiated site was then grafted with 200ul of fresh human lipoaspirate. Graft retention was measured radiographically over the subsequent 8 weeks. The calvarial skin was then harvested for histological assessment of fibrosis as well as for the abundance of En1-positive fibroblasts. RESULTS: FACS analysis revealed that >90% of the hematopoietic cells (CD45+) in the peripheral blood ofEn1Cre;R26mTmG mice were membrane tomato (mT-) negative and membrane GFP negative (mG-) 2 weeks after bone marrow transplantation, indicating successful reconstitution (Fig. 1Bi-ii). Scalp irradiation induced a significant expansion of the GFP+ profibrotic En1-positive fibroblasts 4 weeks following scalp irradiation (Fig. 1Ci-ii). While reduced volume retention of grafted fat was observed over 8 weeks (Fig. Di-ii), this was associated with significant remodelling of the overlying skin; histological evidence of decreased collagen as well as a reduction in the profibrotic En1-positive fibroblast subpopulations was observed compared to irradiated but not grafted sites (Fig. 1E). CONCLUSION: Here, we show that fat grafting reduces radiation-induced scalp skin fibrosis by reducing the skin collagen content, remodelling collagen fiber networks, and by altering the composition of dermal fibroblast subpopulations. Specifically, we show that fat grafting reduces the abundance of the profibrotic En1-positive fibroblasts. Greater exploration of how radiation alters the function of this fibroblast subpopulation may lead to refinedtherapies for patients suffering from radiation-induced tissue fibrosis.
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