Repair and grafting fat and adipose tissue

Adipose tissue: structure and physiology

Adipose tissue is primarily comprised of large lipid-laden adipocytes, which are surrounded by various stromal vascular cells, each with a unique role. Stromal vascular cells include fibroblasts, immune cells, pericytes, and endothelial cells. The extracellular matrix that interconnects adipocytes and forms the fat lobules within adipose tissue provides the structural framework of adipose tissue. There are two general types of adipose tissue: brown fat and white fat. In humans, brown adipose tissue is predominantly found during the neonatal period and is responsible for generating thermogenesis from triglycerides. Brown fat deposits do not appear to play a significant role in human adult metabolism, although recent research has begun to emerge on the importance of brown fat. The discussion here will be limited to the role of white fat, as it is used exclusively for fat grafting procedures.

White fat is involved in a variety of physiologic roles, including the storage of energy-rich triglycerides, cushioning of vital structures and organs, maintenance of metabolic homeostasis, immune regulation, reproduction, and angiogenesis. The imbalance of adipose tissue resulting in either too much fat, such as in generalized obesity, or too little fat, such as in genetic or acquired lipodystrophies and aging, is an increasingly prevalent problem worldwide. Associated disorders are generally linked with physiologic derangements in insulin metabolism, triglyceride and cholesterol stores, as well as generalized insults to end organs involved in these pathways. The increased recognition of these problems has highlighted the need for a more complete understanding of adipose biology.

Adipose tissue influences metabolic homeostasis through production of assorted hormones, cytokines, growth factors, and other peptides. Factors secreted by adipose tissue are involved in a broad spectrum of physiological processes and molecular pathways, including: lipid and steroid metabolism, growth factor signaling, protein binding, cytokine signal transduction, vasoactivity, eicosanoid activity, alternative complement system activation, and extracellular matrix deposition. These effector molecules, termed adipokines, exert their influences in endocrine, paracrine, and autocrine manners. Major adipokines leptin and adiponectin are implicated in obesity and function by regulating appetite and energy expenditure. Tumor necrosis factor–alpha (TNF-α), interleukin-8 (IL-8), and interleukin-6 (IL-6) are all increased in obesity; they function as proinflammatory cytokines promoting increased insulin resistance. In addition, type 1 plasminogen activator inhibitor (PAI-1) is also increased in obesity and functions to promote thrombosis by inhibiting fibrinolysis, acting as a main endogenous regulator of the coagulation system.

Adipocyte differentiation begins with the transformation of mesenchymal stem cells into adipoblasts, preadipocytes and, finally, mature lipid-synthesizing, lipid-storing adipocytes. Preadipocytes bear striking resemblance to early fibroblasts in terms of their cellular architecture and cytoskeletal arrangement. Differentiation involves the coordinated expression of specific genes involved in producing the adipocyte phenotype. Transcriptional regulation of adipocyte differentiation during development is largely controlled by peroxisome proliferator-activated receptor-γ (PPAR-γ). PPAR-γ is the factor demonstrated to be the most specific to adipogenic differentiation. When its receptor becomes activated by its agonist ligand in a target cell, a process of differentiation into an adipocyte begins via morphologic changes, lipid accumulation, and activation of genes distinctively expressed by the mature adipocyte. Additional support during differentiation comes from CCAAT/enhancer-binding proteins (C/EBPs), with C/EBP-β and C/EBP-α specifically stimulating PPAR-γ expression during early differentiation and C/EBP-α having a similar role later in the pathway. Recently, lipoprotein lipase (LPL), specific Krüppel-like factors (KLF5, KLF15, and KLF2), early growth response 2 (Krox20), and early B-cell (O/E-1) factors have also been implicated in adipocyte differentiation. Both insulin and insulin-like growth factor-1 (IGF-1) are known to be required for adipocyte differentiation. Simultaneously, preadipocyte factor-1 (pref-1), whose role is in the maintenance of the preadipocyte phenotype, is decreased during adipocyte differentiation. Late markers of differentiation, which are factors produced once adipocytes mature, include adipsin, angiotensinogen II, acyl-coenzyme A (Co-A)-binding protein (ACBP), leptin, and two fatty acid-binding proteins (FABP) known as adipocyte lipid-binding protein (aP2) and keratinocyte lipid-binding protein. As preadipocytes differentiate into adipocytes, they lose their morphologic similarities to fibroblasts via decreased expression of collagen types I and III and increased production of collagen type IV, laminin, entactin, and glycosaminoglycans. In fact, inhibition of collagen synthesis during this phase actually blocks preadipocyte differentiation.

Biology of ASCs

Preadipocytes were first described in 1976 by Dardick in a rat model; similar cells were later isolated from human adipose tissues. Isolated preadipocytes were initially used to probe adipocyte biology in vitro , leading to the recognition that different anatomic locations and adipose depots express different biological characteristics, such as adipocyte size and lipolytic potential. In 2001, Zuk first discussed the differentiation plasticity of preadipocytes and eventually the term adipose-derived stem cell (ASC) was adopted to encompass the characteristics of self-renewal, asymmetric division, and multi-lineage potency. Although ASCs proliferate and rapidly expand in culture, exogenous growth factors are required to induce lineage-specific differentiation. Differentiation of ASCs to other mesenchymal phenotypes has been well established both in vitro and in vivo . ASC differentiation to cell lineages of the ectodermal and endodermal germ layers has also been successful in several studies.

Adipose-derived stem cells (ASCs) are prevalent within human adipose tissue, surrounding blood vessels and residing within the connective tissue framework. These non-lipid-laden stromal cells are easily isolated from either suction-aspirated adipose tissue or excised human fat via enzymatic collagenase digestion. Numerous descriptions for cell isolation methods appear in the literature. The resultant freshly isolated cell pellet is highly heterogeneous and is named the stromal vascular fraction (SVF). When SVF cells are placed in culture, the ASCs adhere to the surface of an untreated tissue culture flask after 6–8 hours' incubation at 37°C and 5% CO ₂ . Once ASCs have adhered to the culture flask surface, non-adherent populations, representing approximately 7–15% of the SVF and consisting of mainly hematopoietic origin cells, are washed away with sterile phosphate buffered solution and/or fresh culture media. A commonly used ASC expansion medium consists of a Dulbecco's Modified Eagle Medium (DMEM) and DMEM/F12 media combination, with 10% serum, antibiotic (e.g., penicillin, streptomycin), and a small amount of dexamethasone to halt differentiation to another mesenchymal lineage. Once in culture, specific growth factors or other additives can be applied to direct the differentiation to a specific phenotype, such as adipose, bone, cartilage, or muscle.

SVF is attractive therapeutically because it may be obtained from tissue within 60–90 minutes, and the isolation can be performed in a clean room near an operating room, or even within an operating room using available automated devices. Collagenase digestion results in approximately 2–5 × 10 ⁵ nucleated SVF cells per gram of adipose tissue. However, complete ASC isolation takes 24 hours and requires access to cell culture facilities. Benefits of cell culture also include the ability to differentially expand the cell number, select for specific subpopulations, or control the microenvironment for directed differentiation, or seeding of a scaffold material. Flow cytometry characterization of the surface markers on freshly isolated and cultured adipose-derived cells can be performed, and shows the presence of early progenitor markers such as CD34 and CD90.

Freshly isolated SVF obtained directly from harvested adipose tissue has diverse patterns of cell surface markers as identified by flow cytometry. Zimmerlin and colleagues identified several similar cell surface markers in ASCs compared with bone marrow-derived stem cells (BMSCs). Li and colleagues reported four subpopulations of interest that can be isolated and cultured. The first subpopulation is CD31 ⁺ /CD34 ⁻ and is classified as “mature endothelial” as it expresses the endothelial marker of CD31 but lacks the progenitor marker CD34. The second subpopulation is classified as “endothelial stem” and is CD31 ⁺ /CD34 ⁺ , expressing both endothelial and progenitor markers. A third subpopulation, CD31 ⁻ /CD34 ⁺ is classified as “adipose stem” (i.e., ASC), displaying just progenitor markers. The fourth subpopulation represents a “pericyte group” characterized by surface markers CD146+/CD90 ⁺ /CD31 ⁻ /CD34 ⁻ . Pericytes function as contractile cells that regulate blood flow and reside adjacent to the blood vessel walls as shown by immunostaining. Similar to BMSCs, ASCs do not express major histocompatibility complex (MHC)-II and inhibit proliferation of activated peripheral blood mononuclear cells, suggesting a role for modulating the immune system in inflammatory disorders or allogeneic transplantation. In fact, ASCs are an active target of much research in the area of vascularized composite tissue transplantation, hoping to aid in the induction of tolerance to transplanted allograft tissues.

Non-enzymatic isolation of ASCs and SVF has been a topic of recent interest, driven by a potentially less restrictive regulatory pathway. Strategies have focused on mechanical forces such as ultrasound, but there is no evidence to suggest equivalence to enzymatic digestion. There has also been interest in whether there are viable ASCs in the aqueous portion of the lipoaspirate, obviating the need to expose tissue grafts to digestive enzymes. Although some cells are present, the quantity is insufficient for clinical use. Because the ASCs are firmly embedded within the connective tissue, enzymatic digestion is currently necessary to release them in significant quantity for use in clinical applications.