Model of Radiation-induced Hind Limb Contracture and Skin Fibrosis Rescued by Fat Grafting

Abstract

INTRODUCTION: Radiotherapy is an effective anti-cancer treatment, able to reduce tumor size and decrease local cancer recurrence. However, the long-term outcome of radiotherapy is significant and pathologic fibrosis of the soft tissue surrounding the malignancy. Radiation-induced soft tissue fibrosis can disrupt tissue esthetic appears and impair function, such as impaired swallowing and limb contracture. Fat grafting is gaining popularity as a surgical technique able to prevent or reverse the radiation-induced soft tissue fibrosis. We developed a mouse model of radiation-induced hind limb contracture and investigated the potential of grafted fat to restore mobility to the irradiated hind limb. METHODS: The hind limbs of Prrx1Cre;R26mTmG mice were irradiated with 30 Gy fractionated in 5 Gy doses every 2 days for a total of 12 days. The Prrx1Cre;R26mTmG mice were used to label a fibrogenic subpopulation of fibroblasts in ventral skin (PRRX-1+) by embryonic expression Cre. A 4-week period followed irradiation to allow limb contracture to develop, and mice were then sacrificed, and hind limbs were processed for histology. To explore the therapeutic effects of fat grafting, CD-1 nude mice were irradiated with the same irradiation protocol, and at 4 weeks, the mice were injected with 200 μl of human lipoaspirate fat or lipoaspirate enriched with stromal vascular cells (10,000 cells/200 μl) directly into the subcutaneous space on the ventral surface of the irradiated hind limbs. We used 2 control mice; mice injected with 200 μl of saline or mice who received sham surgery with no injection. Limb extension was measured every 2 weeks for a total of 12 weeks, and mice were then sacrificed for hind limb skin mechanical strength testing and histologic analysis. RESULTS: Hind limb irradiation significantly reduced limb extensibility compared to the nonirradiated side, and contracture was associated with a significant increase in the fibrogenic Prrx1+ fibroblast subpopulation in mouse ventral skin. Fat grafting progressively increased limb extension, reduced skin stiffness, and reversed the fibrotic histologic changes in the skin. The greatest improvements were found in mice who received fat grafted with stromal vascular cells. CONCLUSION: We present a mouse model of radiation-induced hind limb contracture which we use to show that grafted fat can reverse the fibrotic changes seen in irradiated skin and can improve the extensibility of contracted limbs postirradiation.