Cd146+ Subpopulation Of Adipose-derived Stromal Cells Augmentation Of Fat Grafts Reduce Soft Tissue Atrophy Caused By Radiotherapy
Nestor M. Diaz Deleon, Mimi R. Borrelli, MBBS, MSc, Sandeep Adem, M.S., Abra H. Shen, S.B., Michael T. Longaker, M.D., M.B.A., Derrick C. Wan, M.D..
Department of Surgery, Division of Plastic Surgery, Hagey Laboratory for Pediatric Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA.
Purpose: Radiation therapy remains a pivotal component for head and neck cancer treatment, but carries adverse side effects that negatively affect tissue function. The most common detrimental sequelae of radiotherapy include contour deformities, skin retractions, restricted movement, and non-healing wounds. Healing via fibrosis activated by radiation heavily damages vascularity of soft tissue, resulting in increased fibrosis and collagen deposition which negatively impacts tissue functionality and elasticity. Autologous fat transfer enriched with adipose-derived stromal cells (ASCs) have been observed to induce a regenerative effect in irradiated tissue by improving vascularity and dermal structure. Previous studies highlight a subpopulation of ASCs, CD146+, which express significantly higher amounts of VEGF, a signaling factor that promotes growth of new blood vessels and improves vascularity. We hypothesized that fat grafts enriched with CD146+ ASCs, compared to CD146- ASCs, will improve skin and fat quality vascularity and fibrosis.
Methods: CD-1 nude immunodeficient mice (n=25) were irradiated at the scalp (30 Gy total) and after 4 weeks, grafted with lipoaspirate and ASCs, which expressed negative hematopoietic markers (CD45, CD235a, CD31) and a positive marker for CD34, isolated via FACS from human patients. The treatment groups were: fat only (n=5), fat enriched with isolated ASCs (n=5), fat enriched with isolated CD146+ ASCs (n=5), fat enriched with CD146- ASCs (n=5), and control saline injection (n=5). MicroCT scans were obtained biweekly for 8 weeks. After 8 weeks of fat grafting, skin and fat grafts were harvested and analyzed by histology. Harvested fat grafts were sectioned and stained with hematoxylin and eosin (H&E) and CD31 immunofluorescence. Harvested mice skin from the scalp was sectioned and stained for Massonís Trichrome, H&E, Picro Sirius Red, and CD31 immunofluorescence.
Results: Fat grafts enriched with CD146+ ASCs were significantly more vascularized than CD146- fat grafts, as exhibited by CD31 immunofluorescence staining. MicroCT scans demonstrated improved fat graft viability after 8 weeks with CD146+ ASC enrichment compared to CD146- ASC enrichment (Figure 1B). Skin histology for mice grafted with CD146+ ASC enriched fat also displayed significantly less dermal thickness and collagen deposition compared to CD1 nude mice grafted with CD146- ASC enriched fat (Figure 1A). Immunofluorescence of skin grafted with fat enriched CD146+ ASCs also revealed significant revascularization after radiotherapy and less collagen deposition.
Conclusions: Fat grafts enriched with ASCs improve fat graft viability and can even vascularize radiation damaged skin. The CD146+ subpopulation of ASCs, which express high amounts of VEGF, can also mediate blood vessel formation when supplemented within fat grafts. This regenerative effect on skin is highly promising for patients with detrimental wound healing impairment and deformities following radiotherapy. Further studies will observe other subpopulations of ASCs that can enhance regenerative effects of fat grafts and bridge the gap of knowledge of ASCs.
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