Strategic Sequences in Fat Graft Survival Jimmy Guo, Alan D. Widgerow, MBBCh, MMed, FCS, FACS, Derek Banyard, MD, MBA, Jason Toranto, MD, Garrett A. Wirth, MD, MS, FACS, Keyianoosh Paydar, MD, FACS, Ilaria Tocco-Tussardi, MD, and Gregory R.D. Evans, MD, FACS Abstract: Although lipotransfer, or fat grafting, is a commonly used procedure in aesthetic and reconstructive surgery, there is still variability in graft survi- val and neoadipogenesis from one procedure to the next. A better understanding of the sequential molecular events occurring with grafting would allow us to strat- egize methods to improve the regenerative potency of the grafted tissue. These steps begin with an autophagic process, followed by the inclusion of stromal vascular fraction and matrix components. By tailoring and modifying each of these steps for a particular type of aesthetic or reconstructive procedure, strategic sequencing represents a dynamic approach to lipotransfer with the aim of maxi- mizing adipocyte viability and growth. In the implementation of the strategic se- quence, it remains important to consider the clinical viability of each step and its compliance with the US Food and Drug Administration regulations. This re- view highlights the basic science behind clinically translatable approaches to supplementing various fat grafting procedures. Key Words: fat graft, adipose-derived stem cells, stromal vascular fraction, progenitor cells, autophagy, adipocytes, endothelial cells, pericytes (Ann Plast Surg 2015;74: 376–382) L ipotransfer (LT), or fat grafting, has become an integral part of the plastic surgeon's armamentarium both in the aesthetic and recon- structive realms. The early, simplistic notion that fat autogenously trans- ferred from one part of the body to another survives and proliferates in its new environment has been superseded by an understanding of pathophysiologic sequelae that occur with the transfer of this tissue. Initially, a complex phase of graft cell death is followed by a replace- ment of new tissue generated by various progenitor cells that interact with their extracellular matrix (ECM). 1 By defining these sequences, certain strategies may be developed that influence or enhance each phase with the aim of improving overall graft survival. To achieve this aim, it is apparent that the components that make up the LT harvest, preparation, and delivery are critical to this process. Furthermore, in the construction of this convention, it is important to consider US Food and Drug Administration (FDA)–compliant procedures to allow for efficient clinical translation. This review describes these essential com- ponents and discusses the possible interventions or influences that com- prise “strategic sequences” in LT. CELLULAR COMPOSITION (EXCLUDING PROGENITOR CELLS) AND ITS IMPACT ON LT Adipose tissue is a type of loose connective tissue that contains an eclectic reservoir of cells including immune cells, erythrocytes, progenitors, and stromal components. 2 Through mechanical processing and enzymatic digestion of the tissue, a stromal vascular fraction (SVF) of cells with therapeutic utility and regenerative potential can be isolated. The SVF represents a convenient source of cells because it is easily obtained and yields a relatively high proportion of stem cells when compared to other methods of mesenchymal stem cell (MSC) iso- lation. Hence, much of the attention relating to SVF has focused on the isolation and culture of multipotent adipose-derived stem cells (ADSCs), a form of MSC that can differentiate along multiple lineages or secrete a wide variety of growth factors. 2,3 Despite this, we believe that the entire composite of SVF cells may be comparable to cultured ADSCs alone in terms of its regenerative potential. Stromal vascular fraction is inherently better suited for clinical applications than ADSCs as it bypasses the extensive time spent in culture. Furthermore, studies suggest that crosstalk between different cell types found in the SVF is mutually beneficial. 4,5 In this section, we explore the interactions among mature SVF cells that constitute an optimal regenerative combi- nation (Table 1). The most common cell type found in adipose tissue is the adipo- cyte. Adipocytes are fat cells characterized by large lipid droplets within the cytoplasm. 6 Among the adipocytes that survive the harvest- ing process during fat grafting procedures, some begin to die on day 1 of transplantation. 1 In the long-term, Kolle et al 7 report high adipocyte resorption rates ranging from 25% to 80%. Despite this, adipocytes remain an important part of the grafting process because their break- down releases recyclable proteins that may be used by neighboring cells in a process known as autophagy. 8 Studies have shown that extensive crosstalk exists between adipo- cytes and macrophages during inflammatory regulation, preadipocyte differentiation, and ECM degradation. 9–12 Adiponectin, a growth factor secreted by adipocytes, causes macrophages to shift from an M1 inflam- matory phenotype to an M2 anti-inflammatory phenotype that is condu- cive to regeneration. 11 More importantly, the co-culture of adipocytes with macrophages spawns an increase in the formation of preadipocytes, the central crux of neoadipogenesis. 10 The macrophage is also a key player in the remodeling of adipose tissue. Macrophages are able to de- grade and constructively remodel biologic scaffolds, most notably the ECM, through their phagocytic functions. 9,12 In the context of LT, we hy- pothesize that SVF macrophages sculpt the ECM into a stromal matrix conducive to angiogenesis and the differentiation of preadipocytes into mature adipocytes. The other immune cells found in the SVF are the T lymphocytes, a group that consists of cytotoxic, helper, and regulatory T cells. 4 Lym- phocytes, like macrophages, are influential in regulating adipose tissue inflammation through mutual cellular crosstalk. 13 The result is a deli- cately balanced milieu that simulates a natural, self-regulating environ- ment for immunomodulation. The stromal component of the SVF includes fibroblasts, 2 a cell type that is integral to the formation of the ECM scaffold in adi- pose tissue. Fibroblasts are elongated, branched cells that secrete col- lagen and other structural proteins to form the framework for its surrounding microenvironment. 14 Beacham et al 15 demonstrated that fibroblasts left in culture produce fibrillar lattices that resemble mesen- chymal matrices and provide a means of attachment for many progeni- tor and stem cells. The presence of collagen-secreting fibroblasts can Received June 12, 2014, and accepted for publication, after revision, November 11, 2014. From the Department of Plastic Surgery, Center for Tissue Engineering, University of California, Irvine, Orange, CA. Conflicts of interest and sources of funding: none declared. Reprints: Alan D. Widgerow, MBBCh, MMed, FCS, FACS, Department of Plastic Surgery, Center for Tissue Engineering, University of California, Irvine, 101 S City Dr, Orange, CA 92868. E-mail: awidgero@uci.edu. Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved. ISSN: 0148-7043/15/7403–0376 DOI: 10.1097/SAP.0000000000000416 REVIEW ARTICLE 376 www.annalsplasticsurgery.com Annals of Plastic Surgery • Volume 74, Number 3, March 2015 Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.