Lipotransfer: the potential from bench to bedside.

REVIEW ARTICLE

Lipotransfer The Potential From Bench to Bedside Ilaria Tocco, MD,*Þ Alan David Widgerow, MBBCh, FACS, FCS, MMed,*þ Shadi Lalezari, MS,þ Derek Banyard, MD, MBA,*þ Ashkaun Shaterian, MD,* and Gregory R.D. Evans, MD, FACS*þ Abstract: The development of autologous fat grafting to augment or reconstruct tissue defects has become an increasingly popular modality among plastic surgeons. Despite its popularity, a standardized fat grafting protocol has yet to be developed. Great variations exist with regard to almost all the technical features, yielding a reported fat graft survivability that ranges from 40% to 80%. Recent bench approaches have been proposed to improve the long-term viability of fat grafts: although promising results have been shown, empirical evidence has yet to prove the superiority of one particular method. Nevertheless, currently available literature still provides some evidence for optimal results in differing clinical scenarios, in the wait of validating and ultimate studies. The issues of enriched fat grafting techniques and variations in harvesting and delivery in the background of US regulatory constraints demand alterations and variations in techniques. These only complicate the process of validation of any single technique. However, recent studies have brought us closer to making informed decisions on technical choices in lipotransfer. These are elaborated on in this review. Key Words: lipotransfer, stromal-vascular fraction, adipose-derived stem cells, centrifuge, decantation, fat graft (Ann Plast Surg 2014;72: 599Y609)

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oft tissue contour deformities result from various causes including cancer, trauma, and chronic wounds, and their reconstruction or restoration remains a challenge in plastic surgery. Approaches to restore soft tissue defects span the reconstructive ladder and include primary closures, grafting, and tissue transfer techniques. The development of autologous fat grafting to augment or reconstruct tissue defects has become an increasingly popular modality among plastic surgeons. A recent survey of plastic surgeons revealed that 57% reported performing more than 10 such procedures each year.1 In addition, the ability to transfer autologous tissue in large amounts without significant donor-site morbidity has made fat grafting an ideal filler in esthetic surgery, with improved short- and long- term patient satisfaction.1 Despite its popularity for soft tissue augmentation, a standardized fat grafting protocol has yet to be developed.1,2 A variety of techniques have been reported in the literature, each with variable results.3Y9 Many of these studies have been criticized for a lack of quantitative evidence supporting fat graft survivability or the Received September 8, 2013, and accepted for publication, after revision, December 30, 2013. From the *Aesthetic and Plastic Surgery Institute, University of California, Irvine, CA; †Clinic of Plastic Surgery, University of Padova, Italy; and ‡Laboratory for Tissue Engineering & Regenerative Medicine, University of California, Irvine, CA. Conflicts of interest and sources of funding: none declared. Reprints: Alan David Widgerow, MBBCh, FACS, FCS, MMed, Aesthetic and Plastic Surgery Institute, University of California, Irvine, 200 S. Manchester Ave, Suite 650, Orange, CA 92868-3298. E-mail: [email protected]. Copyright * 2013 by Lippincott Williams & Wilkins ISSN: 0148-7043/14/7205-0599 DOI: 10.1097/SAP.0000000000000154

Annals of Plastic Surgery

& Volume 72, Number 5, May 2014

predictability of volume restoration.2 As a result, the viability of grafted fat seems to be technique and operator dependent. Variations exist with regard to fat graft handling, harvesting, processing, and reinjecting, yielding a reported fat graft survivability range from 40% to 80%.10Y12 Although various approaches have been proposed to improve the long-term viability of fat grafts, empirical evidence has yet to prove the superiority of one particular method.13Y15 Today, there is no consensus on the best fat grafting technique toward improving fat graft survivability, volume maintenance, and overall graft success. Therefore, the aim of this article was to review current international literature to address the factors implicated in fat graft survival and to devise evidences for optimal results in differing clinical scenarios. Current adipose tissue engineering techniques and the specifics regarding fat graft harvesting, processing, and injecting will also be discussed.

FAT COMPONENTS Adipose tissue derives from the mesodermal layer of the embryo and develops both prenatally and postnatally.16,17 The earliest evidence of adipocytes in humans takes place during the second trimester.18 Macroscopically, 5 different types of adipose tissue have been identified: bone marrow, brown, mammary, mechanical, and white, each serving a distinct biological function. In the bone marrow, adipose tissue occupies space no longer required for hematopoiesis and serves as an energy reservoir and cytokine source for osteogenic and hematopoietic events. Brown adipose tissue is thermogenic, generating heat through the expression of a unique uncoupling protein that short-circuits the mitochondrial pH gradient; it disappears as humans mature. Mammary adipose tissue provides nutrients and energy during lactation and is regulated, in part, by pregnancy-associated hormones. Mechanical adipose depots, such as the retro-orbital and palmar fat pads, provide support to critical structures. White adipose tissue serves to store energy and provide insulation. Although the data are limited, differences in adipose reserves seem to exist with respect to the adipose stem cell expression of fibroblast growth factor (FGF) 2, which is important in angiogenesis and wound healing.19 It remains to be determined as to which human adipose tissue depot should be harvested for optimal stem cell recovery. Since initial reports in the late 1960s,20 multiple independent laboratories have established that stromal cells similar to those identified in the bone marrow can be isolated in a reproducible manner from adipose tissue that is either resected as intact tissue or aspirated using tumescent liposuction.21Y26 Although there is no standard protocol, the adipose tissue is typically harvested, minced, and digested by 1 or more of the following: collagenase, dispase, trypsin, or related enzymes. The released components, defined as the stromal-vascular fraction (SVF), are isolated from the mature adipocytes by differential centrifugation.

Stromal-Vascular Fraction The SVF consists of a heterogeneous mesenchymal population of cells that, in addition to adipose stromal and hematopoietic stem cells, also include endothelial cells, erythrocytes, fibroblasts, www.annalsplasticsurgery.com

Copyright © 2014 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.

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lymphocytes, monocyte/macrophages, and pericytes.23,25Y32 These cells can be seeded in culture media similar to that used for bone marrow mesenchymal stromal cells and purified using a combination of wash and culture expansion steps that act to deplete most of the hematopoietic cell population from the SVF cells. This process allows the emergence of an adherent cell population termed adipose tissueYderived stem/stromal cells.

Adipose-Derived Stem Cells A variety of names have been used to describe the plasticadherent cell population isolated from collagenase digests of adipose tissue. To address the confusion brought among literature, the International Fat Applied Technology Society adopted the term adiposederived stem cells (ADSCs) to identify the isolated, plastic-adherent, multipotent cell population. Adipose-derived stem cells were identified as such by Zuk et al25 in 2001. These authors defined the stem cell characteristics of ADSCs by their ability to differentiate into several mesenchymal lineages (adipocytes, chondrocytes, and osteoblasts, among other lineage pathways).25,33Y42 As stated above, ADSCs can be isolated from the SVF because of their propensity to bind to plastic, unlike the other cell types in the SVF. The third component required to define ADSCs is the presence or the absence of specific cell markers. In particular, ADSCs can be identified by the presence of CD73, CD90, and CD105 and the absence of CD45, CD34, CD14, CD11b, CD79, CD19, and HLA-DR.43 Adipose-derived stem cells have recently been demonstrated, in vitro and in vivo, to possess interesting metabolic properties.44Y53 In particular, ADSCs secrete a favorable cytokine profile that is angiogenic, immunosuppressive, anti-inflammatory, and antioxidative (eg, vascular endothelial growth factor, transforming growth factorYA, hepatocyte growth factor, platelet-derived growth factor, placental growth factor, and basic FGF).35,53Y59

INDICATIONS FOR FAT TRANSFER Fat grafting is indicated for any volume loss due to aging, infection (eg, facial lipoatrophy from human immunodeficiency virus infection), trauma, Parry-Romberg syndrome, scleroderma, hemifacial microsomia, or other causes of soft tissue deficiency resulting in asymmetry or contour irregularity.60 Some clinicians have also used fat grafts for cosmetic breast augmentation.61,62 However, caution is advised in soft tissue defects relating to vascular malformations in which the risk for fat graft oil embolism has been identified.63 Although somewhat controversial, fat grafting has being used for correction of breast lumpectomy defects and for breast reconstruction after cancer ablation.64 More recently, fat grafting has been used for the treatment of radiation damage, breast capsular contracture, damaged vocal cords, and chronic ulceration.65 In 2007, the American Society of Plastic Surgeons Fat Graft Task Force conducted an assessment regarding the safety and the efficacy of autologous fat grafting and concluded that the current recommendation grade for fat grafting is B (meaning that at least fair scientific evidence suggests that the benefits of the clinical service outweigh the potential risks) for breast augmentation and the correction of defects associated with medical conditions and previous breast surgery.66 Other indications including facial augmentation and correction of defects, gluteal and lip augmentation, and hand rejuvenation all received a level 1 grade in the same assessment, meaning that the Task Force was unable to make a recommendation for lack of evidence. Clinical trials for fat grafting are necessary for increasing our scientific understanding and improving the level of recommendation of these procedures.

INDICATIONS FOR ADSC THERAPY Adipose-derived stem cells have the potential for use in regenerative medicine and for the enhancement of fat grafting. In the 600

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United States, SVF-enriched transplantation is primarily used in preclinical research and tissue engineering models because it is believed that more evidence-based medicine is needed to support its use in humans. In other countries, it has been used clinically in human patients to fill large soft tissue defects and for patients with radiation damage or facial lipoatrophy,67 for cosmetic breast augmentation,68 and for treatment of perianal fistulas.69 In vivo studies reveal the potential of ADSCs in rat myocardial infarction models via increased neovascularization, improved wall thickness, and cardiac remodeling.70 In addition, ADSCs have exhibited potential for restoring degenerative disks in a rat model and may prove effective in the treatment of intervertebral disk degeneration by restoration of collagen type 2 and aggregans.71 In mice, ADSC-supplemented fat grafts at 6 months had a greater survival than autologous fat tissue alone.72 In a recent 3-year case study of 30 patients, ADSC-enriched fat grafts were performed for a breadth of cases including polio infection sequel, Parry-Romberg disease, breast reconstruction, scarring, gluteal soft tissue defect, pectus excavatus, and dermatofibromatosis.73 Other indications under study in preclinical animal models include critical-sized craniofacial and long-bone defects, spinal fusion, stroke, spinal cord trauma, multiple sclerosis, Crohn disease, bone marrow transplantation, osteoarthritis, liver regeneration, type 1 diabetes, acute ischemia, wound healing, burns, and tendonitis.74 For lipoinjection, fat is either harvested and finely divided simultaneously, as in suction harvesting, or sequentially harvested and subsequently separated by mechanical means and/or enzymatic digestion. The resulting adipocytes are then reintroduced by some form of injection. The universal principles that underlie successful application of lipoinjection, however, have yet to be determined. Areas of controversy include the following: optimal donor site; technical aspects of graft harvest; the advisability of washing grafts or of otherwise manipulating the harvested fat; the advisability of exposing the graft material to agents such as gravitational force, saline, local anesthetic agents, adrenaline, or buffered salt solutions; appropriate size of the needle for placement of grafts; and the practicality of preservation of graft material for delayed transplantation.

DONOR SITES Over the years, there have been several publications claiming increased efficacy of graft material harvested from one or another donor site.75,76 Most of these reports were based on retrospective observation, and there are few prospective, comparative studies. In terms of cell viability, studies that assessed and compared cells harvested from multiple donor sites did find a similar number of viable preadipocytes from all the clinically relevant donor sites commonly used by plastic surgeons (abdomen, breast, and buttocks).77,78 Only Padoin et al79 reported increased adipose stem cell levels in fat from the lower abdomen compared with that from other sampled anatomical locations.

HARVESTING TECHNIQUES Wet Versus Dry Autologous fat grafting is a technique that dates back to 1893,80 but 100 years passed before the first reported clinical trial.81 During this time, the role of a tumescent solution for hydrodissecting (the ‘‘wet technique’’) and enlarging the target fat layer was emphasized because it was believed to facilitate the subsequent aspiration with decreased pain and ecchymosis. This concept found further confirmation in the 1990s, when studies showed that the so-called wet technique improves the safety of large-volume liposuction by eliminating the need for general anesthesia, intravenous sedation, and narcotic analgesia and results in the reduction of surgical hemorrhage.82 Nevertheless, other authors progressively introduced an alternative ‘‘dry technique,’’ or liposuction performed without infiltration solution * 2013 Lippincott Williams & Wilkins




under general anesthesia and regional blocks.83 This method is highlighted by several advantages, such as a rapid execution and less tissue distortion and damage, as well as disadvantages, such as the requirement for volume resuscitation in large-volume liposuction. In recent years, studies have been conducted to create a datasupported, clinically practical regimen to systematically evaluate the compositional effects of the wet and dry techniques. The findings of the study suggested that the fat collected through the wet technique is more preserved than that collected via the dry technique; however, the results were not statistically significant.84Y86 There have been studies that specifically address the effect of local anesthesia and anesthetic infiltration on human fat viability. These reports suggest that there is no effect of local anesthesia or epinephrine on fat grafts87Y89 but that some local anesthetic agents (lidocaine, ropivacaine, and prilocaine) may modulate isolated preadipocyte viability rates.90,91

Machine Versus Syringe The role of the harvesting technique on fat graft survival is an issue that has been recently addressed by several authors. Because of naivety, it took time for clinicians to realize that a traumatic processing of the lipoaspirate would decrease its longevity.92 By the early 1990s, more positive reports of fat grafting were published, showing improvements in skin and tissue quality, scar revision healing, and volume improvement.93 Nowadays, fat grafts are typically harvested via the syringe aspiration technique with a long atraumatic cannula. Usual fat depot sites include the lower abdomen, the f lanks, and the inner or outer thighs, but other regions including gluteal sites have been used. Coleman94,95 popularized and systematized the ‘‘structural fat grafting’’ technique through the following different concepts: & Harvest of atraumatic small-diameter fat cylinder, by performing a manual aspiration with a 2-hole blunt-tipped cannula, and low negative pressure with a 10-mL syringe & Tissue processing to obtain a purified graft that is free of impurities (centrifugation at 3000 rpm for 3 minutes) & An injection technique that preserves the graft structure without damaging the receptor tissue: fat injection when the 17- to 18-gauge cannula is withdrawn, knitting a tridimensional tissue of linear tunnels, and avoiding localized spherical fat deposits. The result is an increase in the exposition area to the surrounding tissue, facilitating the nutrition through imbibition and the revascularization of the graft. These cannulas are small but wide enough to maintain the fat tissue architecture.

Coleman claims that the best result is achieved by injecting a small volume of autologous tissue every time the cannula is withdrawn so that the graft is deposited in different planes. This should ensure that the tissue grafted is surrounded by the major quantity of well-vascularized tissue and achieved correct revascularization and volume. He reported a variety of more than 1000 patients treated in different facial areas with differing pathologies.94Y97 Although handheld syringe extraction is a preferred method of fat harvest for small-volume grafting procedures (eg, facial grafting), the use of machine-powered negative-pressure techniques (suction-assisted liposuction, ultrasound-assisted liposuction, and vacuum-assisted suction technique) is extremely advantageous for large-volume fat grafting cases including breast and buttock augmentation. To date, the body of evidence does not support one harvesting technique above another in terms of preservation of adipose cell viability.98Y103 However, techniques that use low-pressure suction (j25 mm Hg) by means of larger-bore cannulas (eg, traditional Coleman technique, Viafill system [Lipose Corp, Maitland, Fla], or LipiVage system [Genesis Biosystems, Lewisville, Tex]) seem to increase adipocyte viability.104Y107 * 2013 Lippincott Williams & Wilkins

The Potential of Lipotransfer

The relationship between relative centrifugal force expressed in units of gravity (times gravity) and revolutions per minute is as follows: g ¼ ð1:118 105 ÞRS 2

where g is the relative centrifugal force, R is the radius of the rotor in centimeters, and S is the speed of the centrifuge in revolutions per minute. As an example, centrifugation of a sample at 5000 rpm in a microcentrifuge that has a rotor with a radius of 7 cm will deliver a centrifugal force of 1957 g.

PROCESSING The 4 most common processing techniques for fat grafting are decantation; washing; high-speed centrifugation; and cell-supplemented lipotransfer, also called stromal vascular cell (SVC)Ysupplemented lipotransfer. These techniques have been widely investigated to determine which method leads to a higher percentage of graft retention and improved graft viability.

Decantation and Washing Decantation, the separation of fat components under gravity only, is a graft preparation method that maintains the integrity and number of adipocytes.108 However, the histological analysis of decanted grafts has shown low graft volume retention with severe cystic changes, likely due to contamination from infiltration f luids, blood, and cell debris.109,110 Therefore, some authors have suggested removal of contaminants such as blood, remnants of ruptured fat cells, and cellular debris by washing with buffered salt solutions, 5% glucose solution, or saline,111,112 whereas others have advocated centrifugation.6,109,110 Recent research focusing on human fat processing indicated that washed samples trend toward higher viability and vascularity when compared with the decanted and centrifuged (3000 rpm for 3 minutes) grafts.110 It could be hypothesized that gentle washing eliminates most contaminants during the process, preserving and maintaining the quantity, integrity, and viability of the ADSC component of the aspirated adipose tissue, without subjecting the sample to the harsh conditions of centrifugation.

Centrifugation Centrifugation eliminates many of the contaminants that persist with decantation such as oil, blood, and infiltrate. It also compacts the cellular components with minimal residual excess f luid, facilitating the removal of the unwanted free lipid component.113 Despite having fewer contaminants, centrifuged grafts may have poor viability and histologically show areas of fibrosis and calcification. Investigators have hypothesized that the poor viability of grafts prepared by centrifugation is due to deleterious effects on adipose tissue, such as adipose cell death, caused by strong centrifugation, especially when rotation speed is greater than 1145 g.114 Therefore, new protocols recommend soft centrifugation for a short periodVfor example, 400 g for 1 minute preceded by washing.115

Cell-Supplemented Lipotransfer Grafts enriched with SVCs, also known as cell-supplemented grafts, demonstrate higher vascularity and viability compared with other methods of graft preparation.116,117 They also have optimal outcomes for graft retention compared with alternative methods.118 Enrichment of fat with SVC may also accelerate the regeneration process in vivo, further improving outcomes especially in specific cases such as grafting fat to hostile recipient areas that have extensive fibrosis, after undergoing radiation or burn injury. This technique requires a multistep process, appropriate laboratory equipment, and experienced personnel to be successful. Furthermore, regulatory prohibitions in the United States are a major barrier to the application of this method in a clinical setting. www.annalsplasticsurgery.com


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Local anesthesia is most commonly used, but epidural or general anesthesia may be preferred for removal of larger volumes or when multiple sites are used for harvesting. In local anesthesia cases, use 0.5% lidocaine with 1:200,000 epinephrine into the desired sites.

Abdomen, thighs, flanks, and gluteal region; in general, select sites that are convenient for access BMI 9 25: approximately 1,500 mL available from abdomen flanks or posterior, medial, and lateral thighs BMI G 20: both abdomen and thighs Abdominal or medial thigh fat in short supply (prior liposuction, scarcity of body fat), other sites: suprapubic region, the anterior or lateral thighs, the knees, the lower back, the hips, or the sacrum All relevant donor sites share the similar number of viable preadipocytes.

Recommended Technique


Enhanced volume-retention fat transfer Washing

Centrifuge fat at 1,200 g for 3 min from the syringe suction harvest Decant the upper oil layer Eventually soak up the remaining oil using absorbent cottonoids Transfer the fat into a 1-mL Luer-Lok syringe Transfer fat to a 20-mL syringe and wash gently 3 times in the same syringe with the addition of normal saline at a ratio of 1:1 (vol/vol) Samples are allowed to decant for a few minutes before excess saline is removed after each wash Transfer the fat into a 1-mL Luer-Lok syringe

Conde´-Green et al109 (5) Conde´-Green et al110 (5)

Kurita et al108 (5)

Coleman,93Y95 Coleman and Saboeiro,96 Coleman97

Coleman,93Y95 Coleman and Saboeiro,96 Coleman97 Ozsoy et al104 (5) Erdim et al105 (5) Sommer and Sattler119 (5) Lee et al120 (5)

Ersek92 (5)

Shoshani et al88 (5) Kim et al89 (5) Keck et al90 (5) Keck et al91 (5) Coleman93 (5), Coleman154 (5) Klein82 (2) Agostini et al84 (5) Karacalar85 (5) Karacalar and Ozcan86 (5)

Coleman95 (5) Coleman and Saboeiro96 (3) Coleman97 (5) Moore et al87 (5)

Rohrich et al77 (3) (5) Coleman93 (5) Coleman94 (5)

von Heimburg et al78

References (LOE 1Y5)


Processing Standard autologous fat transfer

Tumescent solution (wet Lidocaine (0.05%Y0.1%; maximum dose is 6 mg/kg of body weight) technique under general anesthesia) + Epinephrine (1:1,000,000Y1.5:1,000,000 [0.25Y1.5 mg/L]); the total dosage should not exceed 50 Kg/kg) + Saline (approximately 1Y3 L; the end point is a firm feel of the skin that makes the skin swollen and difficult to grasp). In all situations, approximately 1 mL of solution is infiltrated for every milliliter of fat to be harvested. Tumescent solution is injected through a 22-gauge spinal needle introduced in the subcutaneous space through a small incision. Whenever possible, harvesting sites are accessed through incisions placed in creases, previous scars, stretch marks, or hirsute areas. Wait for approximately 30 min after tumescence for the infiltration fluid to percolate properly and its full pharmacological effects to take effect (‘‘detumescence’’). Aspiration Cannulas: diameter from 1 to 2.5 mm. Avoid 94.5 mm in diameterVthe risk for bleeding and embolism. Use a blunt-tip and dull distal-opening cannula and twist it onto a 10-mL Luer-Lok syringe. After inserting the cannula tip into the donor site, pull back on the syringe plunger to create approximately 1Y2 mL of space in the barrel of the syringe. When filled, the syringe is then disconnected from the cannula, and the barrel filled with 10 mL of harvested material is placed into a centrifuge.

Anesthesia

Harvesting Donor site

Operative Feature

TABLE 1. Evidence-Based Guidelines for Lipoinjection

Tocco et al


* 2013 Lippincott Williams & Wilkins


Isolate the blood/saline phase (È250 mL)Vcentrifuge at 400 g for 10 min Resuspend resulting pellet in NH4Cl for red blood cell lysis for 2Y5 min; centrifuge at 400 g for 10 min Resuspend the pellet in DMEM with 40%Y50% fetal bovine serum, followed by plating the cells and incubation overnight Wash away nonadherent cells and debris with PBS Differential lipoaspirate centrifugation Simultaneous washing with filtration system (Puregraft; Cytori Therapeutics, Inc, San Diego, Calif).

Rapid SVF isolation method:

Leave fat to decant in the same syringe used for harvest for approximately 15 min Decant the upper oil layer Transfer the fat into a 1-mL Luer-Lok syringe Centrifuge fat at 400 g for 1 min from the syringe suction harvest Decant the upper oil layer Eventually soak up the remaining oil using absorbent cottonoids Transfer the fat into a 1-mL Luer-Lok syringe Prepare half of the lipoaspirate as a graft material: centrifuge at 700 g for 3 min from the syringe suction harvest; place the floating adipose portion in a metal jar and place in an ice water bath. Use the other half of the collected liposuction aspirate for isolation of the SVF: Processed lipoaspirate cells: Digest fat with collagenase in PBS for 30 min Centrifuge to isolate cell pellet (800 g for 10 min) Resuspend in ELB and incubate for 5 min Filter and wash pellet 3 times in DMEM Liposuction-aspirated fluid cells: Centrifuge suction fluid (400 g for 10 min); resuspend the pellet in ELB and incubate for 5 min Filter and wash pellet 3 times in DMEM. Add the fresh isolated SVF to the graft material. Mix gently and wait for 10Y15 min for cell adherence to the aspirated fat. Blunt 1-mL cannula injection of small aliquots (injection rate, 0.5Y1 mL/s). Insert subcutaneously in several layers and directions, thereby ensuring diffuse distribution of the graft material. In the face, 7- and 9-cm cannulas are the most useful; longer cannulas, up to 15 cm, can be useful in the body. For varying situations in the face and body, cannulas with different tip shapes, diameters, lengths, and curves can be used.

Francis et al155 (5) Widgerow et al, unpublished data Zhu et al156 (5)

Carpaneda and Ribeiro122 (5) Guerrerosantos et al123 (5) Karacaoglu et al124 (5)

Sherman et al121 (3)

Coleman,93Y95 Coleman and Saboeiro,96 Coleman97 Ozsoy et al,104 Erdim et al105 Sommer and Sattler,119 Lee et al120

Kuroda et al135 (5)

Matsumoto et al134 (5)

Botti et al111 (3) Boschert et al112 (5) Ramon et al6 (5) Conde´-Green et al110 Hoareau et al115 (5)


BMI, body mass index.

Machine-assisted processing

Future directions Alternatives to enzyme digestion

Implantation

Enriched fat lipotransfer

Low-centrifuge technique

Gravity sedimentation

Annals of Plastic Surgery The Potential of Lipotransfer



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INJECTION AND IMPLANTATION TECHNIQUES The method of fat reinjection and implantation after graft harvest has also been evaluated in the current literature, with a specific focus on cannula size, injection volume, and location of implantation.

Cannula Size Ozsoy et al104 compared 3 different diameters of Colemantype injection cannulas and found that adipocyte viability was greater with 2.5-mmYdiameter injection cannulas compared with smaller-diameter cannulas (1.6 or 2 mm). In contrast, Erdim et al105 compared 3 types of needles for reinjection (14, 16, and 20 gauge) and found no significant differences between needle gauge and cell viability. Sommer and Sattler119 also concluded that fat graft survival was less dependent on reinjection methods after comparing needle diameter sizes. Although these studies represent differing conclusions about needle/cannula sizes, experiments by Lee et al120 demonstrated that a slow f low rate (0.5Y1 mL/s) yields 38% more graft take than does a fast injection (3Y5 mL/s), which may explain the potential relationship between cannula size, f low rate, and fat viability. Shear stress is an important mechanical force exerted on adipocytes during cannula fat injection and one that may depend on cannula size and the surgeon.

Injection Volume The volume of fat injection has also been found to be a significant predictor of fat graft survival. Fat grafts initially obtain nutrition through plasmatic imbibition before becoming vascularized, and their survival therefore initially depends on the volumeYsurface area ratio. Studies have found that single-bolus fat injections with high injection volumes can result in poor outcomes and cause significant complications including fat necrosis, blindness, or strokes.121 Carpaneda and Ribeiro122 revealed that the percentage of fat graft viability depends on the thickness and geometric shape of the graft in the recipient bed and suggested an inverse relationship between the percentage of graft survival and the graft diameter. To this end, smaller-volume fat injections across larger grafting diameters will yield improved results.

Injection Plane The tissue plane of the fat grafts after reinjection has also been investigated in the current literature. Many plastic surgeons use techniques consisting of multiple trochar passes with fat deposition across different tissue planes to increase capillary contact and fat graft survival. Although few clinical studies have evaluated the relationship between fat graft survival and reinjection in tissue planes, preclinical models have demonstrated useful data. Authors have found improved survival rates when fat is injected into the subcutaneous,122 intramuscular,123 or supramuscular tissue plane,124 whereas reinjection of fat into the submuscular plane has yielded suboptimal results.124

Fat Storage for Future Use The effect of different fat storage techniques on adipose tissue viability has been investigated by several studies.125Y131 These studies conclude that frozen fat can be used for autologous fat transfer but that the addition of a cryoprotective agent (eg, 10% dimethylsulfoxide, 7.5% polyvinylpyrrolidone 40/7.5% dimethylsulfoxide, 10% glycerol, or 10% glycerol/10% fetal bovine serum) and a strict methodology of freezing (from j40-C down to j80-C) improve subsequent cell viability.

FAT TISSUE ENGINEERING Implementing the Potential of Stem Cell Therapy Several authors have tested the potential benefit of SVF supplementation to improve fat graft survival and maintenance. Stromalvascular fraction can favor neoangiogenic vascularization and fibrogenic 604



activity that enhance adipose tissue survival and 3-dimensional organization. Compared with traditional fat grafting, the survival of the SVF graft is more probable, and fat necrosis is reduced. A clinical data review by Gentile et al132 supports the beneficial effect of the use of SVF mixed with fat graft material for the filling of breast soft tissue defects, observing a 69% maintenance of the contour restoring and of 3-dimensional volume after 1 year compared with 39% of patients in the control group who were treated with centrifuged fat graft. Platelet-enriched plasma is another adjuvant that was found to be successful in its ability to improve vascularity in fat grafting.132 Similarly, Butala et al133 tested the mobilization of endogenous progenitor cells to accelerate the bone marrowYderived cell response to injected fat. The final effect was an enhancement of low-density fat neovascularization, an increase in cytokine expression (SDF-1> and VEGF), and an improvement of graft survival to a level equal to that of high-density fat grafts. Currently, one of the most interesting and promising techniques is the approach to autologous fat grafting called cell-assisted lipotransfer (CAL) for transplantation of aspirated fat tissue and adipose progenitor cells or ADSCs.67,68 Stromal-vascular fraction containing ADSCs is freshly isolated from half of the aspirated fat and recombined with the other half. The freshly isolated SVF used in CAL contains ADSCs as well as vascular endothelial cells, pericytes, blood cells (both white and red), and other cells, as previously described.67,68,134 One hypothesis is that after transplantation, ADSCs may interact with and augment the activity of other cells such as vascular endothelial cells. Ultimately, supplementation with SVF may be superior to ADSCs alone in this treatment; however, further studies are needed to elucidate the synergistic effects of these modalities.134 Finally, the most recent advancement in the field of ADSCbased regenerative therapy is represented by the isolation of a new stem cell population from mesenchymal tissues under cellular stress conditions.135 These cells, termed multilineage differentiating stressenduring (MUSE) cells, are of mesenchymal stem cell origin and exhibit characteristics of both mesenchymal and pluripotent stem cells. Advantageously, MUSE cells do not seem to undergo tumorigenic proliferation and therefore would not be prone to produce teratomas in vivo, nor do they induce immune rejection in the host upon autologous transplantation. In addition, MUSE cells have been shown to home into damaged areas in vivo and spontaneously differentiate into tissue-specific cells according to the microenvironment to contribute to tissue regeneration when infused into the bloodstream. Therefore, MUSE cells exhibit the potential to make critical contributions to tissue regeneration without the restrictions commonly associated with bone marrow stromal cells and human skin fibroblasts, such as time-consuming purification methods and cell sorting.

Adipose Tissue Engineering Techniques Studies have demonstrated novel methods for engineering adipose tissue including (1) scaffold-guided regeneration techniques, (2) injectable composite-based systems, (3) and de novo adipogenesis. Both preadipocytes and adipocytes are anchorage-dependent cells that must be seeded onto an appropriate scaffold surface to provide the traction necessary for cell differentiation and proliferation to occur.136 As such, multiple studies have validated the use of various polymers, including polyester,137Y139 hyaluronic acid,140,141 collagen,136,142 polyethylene glycol,143 and chemically modified alginate144,145 for adipose tissue engineering purpose. To this end, preadipocytes cultured on absorbable polymeric scaffolds and implanted in vivo foster simultaneous cellular proliferation; scaffold resorption; and, ultimately, maturation of adipose tissue. Injectable composite-based systems have also shown promise in creating adipose tissue. This technique comprises seeding preadipocytes * 2013 Lippincott Williams & Wilkins




or adipocytes onto biodegradable beads that are injected in hydrogel carrier medium.136Y146 The hydrogel serves to localize adipocytes in the soft tissue defect by concomitantly filling the tissue defect and supporting adipogenesis. By repeatedly reinjecting this adipocytebiodegradable composite, some investigators were able to fill soft tissue defects of the breast.136Y146 Cell signaling through chemokine and growth factor expression is necessary for the migration and proliferation of adipocytes. An exogenous growth stimulus can therefore stimulate the migration of preadipocytes to the implant site and induce their proliferation and differentiation.147Y150 Studies have found that when matrigel, a collagen basement membrane matrix, was mixed with FGF and injected, it developed into a fat pad secondary to preadipocyte migration and proliferation.147,148 Similarly, investigators have described the development of fat pads after coinjections of photocured styrenated gelatin microspheres and basic FGF, insulin, and insulinlike growth factor I.149,150

DISCUSSION Fat grafting has evolved as a popular method for soft tissue augmentation among plastic surgeons. It has been used for several reconstructive and esthetic purposes,60Y74 and many more applications are currently under evaluation.13,151 For the operative surgeon, there are a large number of technical options for fat harvest and grafting.6,80Y124 Technical adjustments have been developed to enhance the fat graft viability and fat survival to improve patient outcomes. However, the plastic surgery scientific community has yet to develop a standardized procedure protocol, and outcomes from fat grafting have remained largely variable.152,153 Although a variety of different techniques have been independently developed and evaluated in current literature, the conclusions of these numerous studies have historically been difficult to evaluate for the development of a standardized protocol.152,153 Today, a paucity of high-quality clinical studies for any of the identified technical steps in fat grafting has made guidelines in fat grafting difficult to develop. Nonetheless, this review reveals several clear conclusions regarding fat grafting from the current literature and therefore allows the development of ‘‘current practice guidelines’’ according to the tradition of evidence-based clinical medicine (Table 1), which are as follows: & Donor-site selection seems to be unimportant from a cell survival standpoint and can be made at the surgeon’s discretion or on the basis of available donor-site adiposity and/or patient nomination.77,115,119Y124,134,135,154Y156,78,112 Nevertheless, it is a basic tenet of plastic surgery that like should be replaced with like.93Y97 & The decision to use epinephrine/local anesthesia should be based on other clinical factors such as pain relief and bleeding control rather than fat cell viability.87Y91 & There is no significant difference between using the wet or dry technique for autologous fat grafting, and either method can be used reliably with favorable results.84 & Machine-assisted liposuction is a safe fat harvesting technique and comparable with the withdrawal of approximately 1 to 2 mL of fat in a 10-mL syringe.98Y103 & There is no evidence to support the superiority of one processing technique over another.152,153 Different protocols will result in the following diverse qualities of fat: & Centrifugation and gravity sedimentation remove 50% to 60% of red and white blood cells. & Washing with filtration removes more than 95% of both blood cell types; there is no reported difference in the expression levels of growth factors when comparing techniques6,108Y114 and therefore different outcomes (washing results in high viability). * 2013 Lippincott Williams & Wilkins


& High-speed centrifugation results in consistent volume retention but lower viability. & Cell-supplemented lipotransfer has optimal outcomes for graft retention. & When centrifugation is used, forces greater than 1200 g cause more cellular damage.115 & If aspirated fat is to be used, it must be aspirated gently, using a blunt cannula and a 10-mL syringe.93Y97 & There is little consensus on the importance of needle/cannula size for fat graft survival. However, a slow injection rateV0.5 to 1 mL/sVis important for graft take.104,105,119Y124 & Injection volume and injection plane are important factors in graft survival. Injection must be undertaken via a fine cannula and a 1-mL syringe with multiple passes, injecting only a tiny amount with each pass and injecting as the needle is withdrawn.93Y97

SPECIAL TOPIC: LEGAL CONSIDERATIONS IN LIPOTRANSFER United States Human cells and tissues intended for human transplantation are regulated by the Food and Drug Administration (FDA). The FDA maintains 2 levels of classifications for cells and tissues: (1) HCT/P 361 and (2) HCT/P 351. Category 361 is summarized as ‘‘tissue’’ and procedures that take place in the same operative session. These procedures fall under the jurisdiction of practice of medicine. Surgeons follow guidelines and laws established by state medical boards and their professional societies but are not controlled by the FDA. Category 351 is the ‘‘drug/biologic’’ category, which is completely regulated by the FDA.157,158 Recently asked for a statement of position on the matter, the FDA declared to consider autologous adipose stem cells from SVF for reconstructive purposes as a drug because of the use of collagenase for isolation of ADSCs.159 The practical implication is the need for any surgeon who wishes to use the SVF to submit an Investigational New Drug application to the FDA and have an approved institutional review board with the referring institution. Given the time consumption and cost of the overall process, we tend to believe that a physician in the United States will be discouraged to perform the cell-supplemented lipotransfer technique. Furthermore, automated devices for separating adipose stem cells are regulated as class III medical devices by the FDA, and currently, no such device is approved for human use in the United States. Therefore, we advise practitioners to seek for the best fat graft quality by following the general indications stated above. Above all, processing seems to be the most controversial point in terms of definition of the best standard or practice. The principal techniques reported by the literature are all valuable to perform lipotransfer and should be selected according to the aim in a case-by-case manner. Among centrifugation procedures, the most convincing results so far have been provided by the ‘‘low-centrifuge’’ (400 g for 1 minute) technique115 in terms of cell viability and graft survival.

Outside United States In Europe, ADSCs are considered advanced-therapy medicinal products, as defined by the European Regulation (European Commission) 1394/2007, which contains rules for authorization, supervision, and technical requirements regarding the summary of product characteristics, labeling, and the package leaf let of advancedtherapy medicinal products that are prepared following industrial methods and in academic institutions.160 This regulation refers to the European good manufacturing process rules.161 The conversion of research-based protocols using ADSCs into a safe manufacturing process requires protocols that have had careful consideration of all the risks and benefits for the patient and user. That said, the absence of formal prohibitions for the use of enriched-fat lipotransfer in www.annalsplasticsurgery.com


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clinical settings results in a general recommendation for the use of the CAL protocol: this technique has been demonstrated to provide the most satisfying results in terms of long-term outcome, most likely because of the dramatic release of angiogenic growth factors67,68,134

23. Gimble JM, Katz AJ, Bunnell BA. Adipose-derived stem cells for regenerative medicine. Circ Res. 2007;100:1249Y1260.

CONCLUSIONS

24. Young HE, Mancini ML, Wright RP, et al. Mesenchymal stem cells reside within the connective tissues of many organs. Dev Dyn. 1995;202:137Y144. 25. Zuk PA, Zhu M, Mizumo H, et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng. 2001;7:211Y228. 26. Meruane M, Rojas M. Adipose tissue derived stem cells. Int J Morphol. 2010;28:879Y889.

Nowadays, fat grafting has achieved the status of a widely performed and worldwide-performed procedure among plastic surgeons. Nevertheless, it is still in the stage of technical evolution. Surgeons are committed to implement new techniques, and scientists are called to provide alternatives and improvements to the current clinical practice to optimize the operative features of lipotransfer and therefore to improve the clinical outcomes.

27. Cawthorn WP, Scheller EL, MacDougald OA. Adipose tissue stem cells meet preadipocyte commitment: going back to the future. J Lipid Res. 2012; 53:227Y246.

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Lipotransfer: the potential from bench to bedside.

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