It is generally acknowledged that tissue augmentation. Enhancement of Autologous Fat Transplantation With Platelet Rich Plasma ORIGINAL ARTICLES

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1 The American Journal of Cosmetic Surgery Vol. 18, No. 2, ORIGINAL ARTICLES Enhancement of Autologous Fat Transplantation With Platelet Rich Plasma Patrick Z. Abuzeni, MD, DDS; Robert W. Alexander, MD, DMD Introduction: Adipose tissue is readily available for autotransplantation. Over many years, the popularity of fat transplantation surgery has waxed and waned as a result of relatively inconsistent and unpredictable survival. Many factors influence the success of autologous fat tissue grafts, some of which can be controlled by the surgeon. Examples include the use of minimally traumatic cannulae, low pressure suction, careful handling of graft tissues, and aseptic techniques. There is emerging evidence that fat grafts may be made more reliable and consistent by careful selection of donor sites and by influencing certain healing mechanisms that control cellular recruitment, migration, and differentiation at the recipient site. Materials and Methods: This paper presents an innovative technique that provides a means to isolate autologous platelet rich plasma for use with autologous fat for the purposes of enhanced tissue augmentation. Results: The apparent concentration and greater quantity of cellular grafts observed after utilization of these techniques seem to yield a higher proportion of graft volume retention. Discussion: This technique is intended to promote or accelerate the healing phase after grafting, enhance the intended augmentation retention volume, potentially reduce secondary calcifications and microcyst formation, and maximize the transplant unit volume by reducing the extracellular fluids transferred with the grafts. It is generally acknowledged that tissue augmentation with autologous grafts remains the premiere choice for most tissue augmentation and transplantation surgery. It is well established that autologous tissue grafts survive the transfer procedures to eventually survive in the recipient site by the principles of induction and conduction. 1 7 The ideal graft tissue should be readily available and have low antigenicity and donor site morbidity, reproducible retention and results, and a lack of disease transmission (Figure 1). Autologous fat grafting meets all of these criteria Received for publication January 7, Dr Abuzeni is in private practice in Coral Gables, Fla. Dr Alexander is in private practice in Seattle, Wash. Address correspondence to Patrick Z. Abuzeni, MD, DDS, 248 Palermo Avenue, Coral Gables, FL and therefore represents a readily available resource for tissue augmentation. Fat graft survival, although well documented, has been described as being somewhat unpredictable, 4,5,8 11 perhaps because of the lack of consistent harvest, handling, and transfer protocols. Therefore, evaluation of the results of one study is often difficult to reproduce or assess with other experiences in the grafting of fat tissues unless the exact same protocol is utilized. Standardization of such important aspects of autologous fat grafting is evolving at this time. Consistent techniques will facilitate the comparison of ongoing clinical trials to determine the safety and efficacy of such grafts. Some of the factors that appear to exert substantial influence on the success of autologous fat transplantation are overall patient health, genetic predisposition for fat storage in donor sites, pretransplantation and posttransplantation diet, basic metabolic rate, atraumatic harvest and handling, proper preparation of recipient bed, and graft immobilization in the recipient sites (Figure 2). This project was designed to establish a simple and reliable protocol to isolate platelet rich plasma (PRP), which is rich in a variety of potentially valuable growth factors. Growth factors are characterized as regulatory peptides with specific tissue site receptors They serve to regulate and deregulate cellular activity, enzymes, angiogenic factors, antiangiogenic factors, inducers of gene expression, and much more. 14,16 18 By standardization of isolation procedures to collect autologous growth factors for addition to harvested tissues, study of the efficacy and value of such additives can be made. This paper will present a means to effectively isolate and incorporate use of autologous PRP with plateletderived growth factors (PDGF) to study the potential to enhance viability and clinical success of using transplanted fat as a soft tissue augmentation alternative. It is hypothesized that introduction of such growth factors during the preparation and transfer phase may potentiate wound healing through mechanisms that control cellular recruitment, migration, and differentiation at the recipient site. We will provide a brief review of the roles established for these elements. Biology of Wound Healing The clinical value of PRP as a promoter of wound healing is well documented. 15,18 23 The authors believe

2 60 The American Journal of Cosmetic Surgery Vol. 18, No. 2, 2001 Figure 1. Characteristics of the ideal soft tissue graft. that PRP, calcium chloride, and thrombin may increase the retention of the transplanted fat cells and increase the rate of revascularization and survival of the transplanted cells. This enhancement of healing rate and graft acceptance may, in turn, decrease the potential of liponecrosis and microcyst formation. PRP is rich in both PDGF and transforming growth factor- 1 (TGF- 1), which are derived from platelets that contain the highest concentrations of those elements. These growth factors are known to be important in initiation and progression of wound healing. The addition of PDGF has been shown to stimulate the growth of the in vitro cultured cell 18 and to synergistically promote the healing of wounds 15 by its ability to restore the plasma s function. It is also well established that cytokines are intrinsically involved in the healing process. Cytokines implicated in the wound healing process are listed in Figure 3. It appears that PDGF and TGF- 1 are among the most important growth factors in wound healing. Both are present in the alpha granule of the platelet and are released with cell activation and degranulation. Each has shown a marked beneficial effect on wound healing. 30,31 PDGF appears to have a direct mitogenic influence on the target cells by binding to cell surface receptors and by indirectly enhancing the proliferative response of cells lacking detectable PDGF receptors. 15,20 Platelets are the richest known source of PDGF, although it has also been identified within macrophages, fibroblasts, and endothelial cells. 23 TGF- 1 is also found in the platelet alpha granule and in macrophages. TGF- 1 is chemotactic for macrophages and fibroblasts and is well established to be a potent stimulator of granulation tissue formation. Working synergistically, PDGF initially acts as an attractant for monocytes or macrophages, or both, and becomes an activator, causing those cells to start production and secretion of additional PDGF into the wound area, thus establishing an autocrine amplification system. 15 In the case of autologous fat cell transfer, placement of such cells into created potential space (pretunneling) that is surrounded by similar cellular elements with some degree of clotted blood containing platelets may have great clinical importance. The wound potential space and the transplanted cellular elements are initially hypoxic (partial pressure of oxygen of 5 10 mm Hg) and acidotic (ph 4 6). The site contains platelets, leukocytes, red blood cells, and fibrin in a complex clot adjacent to the transferred adipocytes, interstitial collagen, and fibroblasts. Surrounding the graft, the undisturbed recipient tissues are normoxic (partial pressure of oxygen of mm Hg) and maintain a physiologic ph (ph 7.42). This area serves as a resource to the grafted area because it contains a population of structural cells, healing capable cells, and in- Figure 2. Host and perioperative considerations that will influence the success of an autologous fat graft.

3 The American Journal of Cosmetic Surgery Vol. 18, No. 2, Figure 3. List of some of the most common cytokines implicated in wound healing. tact capillaries. At the junction of intact tissue to graft, small capillaries develop terminal clots and offer exposed endothelial cells. This complex environment, simplified for the sake of this discussion, represents a basic model of wound healing of any tissue injury. This endogenous system of repair starts, maintains, and promotes repair related to injury and offers an opportunity for surgeons to promote enhanced tissue regeneration and graft acceptance. The established oxygen tension potential between the wound and the surrounding normoxic tissue bed is capable of inducing macrophage recruitment and promoting angiogenesis by secreting macrophage-derived angiogenesis factor and macrophage derived growth factor. 24,26 The angiogenic effect of macrophages is selectively induced by hypoxia and may be potentiated by hyperbaric oxygen therapy. In the presence of normal oxygen tensions, the number of fibroblasts, amount of collagen deposit, as well as the number of capillaries can also be increased by increasing the number of macrophages present at the wound site. Therefore, it can be concluded that the healing potential of a wound is not only influenced by the presence or absence of chemotactic agents but also by the number of competent cells Platelet gel offers a unique autologous biologic sealant created from PRP, calcium chloride, and thrombin that has been used extensively in a variety of disciplines with remarkable clinical success. 5 7,11,15,19,20,29 Platelet gel in cosmetic surgery may serve primarily as an adjunctive hemostatic agent and graft carrier to a recipient site. Fat Graft Healing Model Some investigators suggest that transplanted mature adipocytes might de-differentiate into a precursor phenotype or maintain mature adipose tissue and survive the transfer. Jones et al 30 clearly demonstrated in tissue culture (in vitro) that mature adipocytes survive and become metabolically active and that no apparent dedifferentiation resulted. In fact, it was suggested that some induction of pluripotential cells may have actually differentiated into mature adipocyte form and initiated liponeogenesis in tissue culture. In the autologous fat graft, we characterize 3 stages of healing (see Figure 4, A and B). During the first stage, cellular differentiation and activation of preadipocytes begins with release of PDGF and TGF- 1 during degranulation of platelets. PDGF is believed to stimulate mitogenesis and differentiation of the preadipocyte. Angiogenesis is simultaneously stimulated, resulting in capillary budding and ingrowth via induction of endothelial cell mitosis. This initial stage of wound healing is measured in days, and activity usually persists for up to 1 week. The second stage, typified by fibroblast activation, usually lasts for up to 2 weeks. It is suggested that TGF- 1 is responsible for initial fibroblast activation, resulting in increased numbers, and stimulates additional differentiation of precursor adipocyte cells. The continued presence of TGF- 1 will further act to stimulate adipocyte lipogenesis while the fibroblasts begin to synthesize procollagen type 1 in preparation for deposit of collagen in the wound site and to provide support for capillary ingrowth. The activated fibroblasts begin synthesis of fibronectin and hyaluronic acid known essential elements of the new extracellular matrix. The third stage is characterized by wound maturation and typically persists for up to 1 year. During this time, TGF- 1 continues to encourage additional fibroblast recruitment and activation while PDGF activates secretion of enzymes (ie, collagenase) to assist in collagen remodeling and wound maturation. The action of these growth factors is clearly synergistic in promotion of healing in wounds. 15 As discussed previously in this paper, PDGF participates in development of the autocrine amplification system. This feedback system both initiates and maintains the healing and integration of the newly transplanted cells by capillary ingrowth and collagen remodeling. Materials and Methods Sequestration of Platelet Rich Plasma The isolation of PRP is accomplished using the Medtronics AT500 gradient density cell separator used

4 62 The American Journal of Cosmetic Surgery Vol. 18, No. 2, 2001 Figure 4. (Top) Stages of wound healing and duration of action. (Bottom) A schematic simplification of the 3 phases of wound healing. Sustained and long-term effects of platelet-derived growth factors are maintained with recruitment and migration of cells that further release platelet-derived growth factors at the surgical site. simultaneously in the operating room during harvest of autologous fat for transplantation. Autologous whole blood ( ml) is withdrawn through a dedicated large-bore peripheral intravenous catheter. The collected whole-blood specimen is placed in a sterile collection bag containing citrate phosphate dextrose as an anticoagulant. The blood is then centrifuged at 5600 rpm to separate the specimen into its 3 basic components: red blood cells, PRP, and the platelet poor plasma layer (PPP). The different densities of these components result in 3 different layers: the blood cell layer on the bottom, PRP layer in the middle, and the lowest density PPP on the surface. The cell separator allows removal of the least dense PPP layer (about 200 ml). The bag is then recentrifuged at a speed of 2400 rpm to allow for precise separation of the PRP layer (about 70 ml) from the denser cellular elements. The PRP is straw colored and will have a red tint when the top 1 mm of the cellular layer is included during removal of the middle layer. The top of the blood cell layer is known to contain most of the recently synthesized platelets that have the greatest activity potential. 3,15 The isolated PRP can then be introduced to the surgical field in preparation for its addition to the harvested and rinsed autologous fat cells while the blood cells and PPP are returned to the patient. The procedure takes approximately minutes and is typically accomplished during the fat harvesting and rins-

5 The American Journal of Cosmetic Surgery Vol. 18, No. 2, ing phase. This service can be provided by local hospital or blood-bank agencies available in most metropolitan areas. Medtronics gradient density cell separators are common appliances in most hospital operating rooms. Closed Syringe Harvest of Donor Adipocytes Adipocytes are harvested using the usual tumescent fluid volume infiltration (fluid to graft ratio of 2:1) composed of normal saline, Klein solution, or lactated Ringer solution and a minimally traumatic technique. We believe that the use of larger diameter blunt cannulae, devoid of air within the system, and the use of limited vacuum pressures combine to minimize the injury to the fat tissues. Fat grafts are collected as atraumatically as possible by use of the patented Tulip Syringe System that applies minimal extraction vacuum pressures. New technology offered by the Tulip company delivers a superpolished titanium cannula system specifically developed and designed for tumescent infiltration and adipocyte withdrawal, avoiding much of the potential damage sustained by contact of fat-cell membranes to the rough stainless steel surfaces of standard cannulae. Syringe-harvested fat should not be compared with that harvested utilizing machine vacuum pump and trap systems. All air is displaced from the syringe and harvesting cannula with sterile saline, thereby completely eliminating air from the system. The importance of this fact may be best appreciated when considering that the rapid rise and fall of pressure gradients associated with machine vacuum systems does cause cavitation and cellular vaporization, resulting in the damage of cells near the harvesting cannula tip. A simple analogy to this can be made in observation of the rapid changes of pressures produced in a household vacuum cleaner being moved between a linoleum floor and deep pile carpeting. The pressure difference developed is capable of creating a cavitational effect in the tissues, resulting in increased cellular trauma. 31 The collected fat is carefully and serially rinsed in sterile saline solution in order to reduce the intracellular lidocaine and debris (including cellular remnants, blood products, free lipids, etc). It is common to perform at least 3 rinses in order to produce the cleanest graft material possible. 32 On completion of this stage of preparation for grafting, additive materials such as PRP may be added to enhance the tissue acceptance and survival of the grafts. Fat Graft Preparation With PRP-Platelet Gel After atraumatic harvest and serial rinsing, fat graft preparation begins with initiation of the coagulation process with PRP. This represents a modification of what has been previously reported in other applications of PRP utilization. 1,3,15,33 PRP is first added to the rinsed fat graft at a ratio of 1:10, and after gentle agitation, it is left undisturbed for at least 10 minutes. There is evidence that the process of growth factor binding to specific receptor sites on the human adipocyte may continue for up to 4 hours. 12 Bovine thrombin ( units) is constituted in 10 ml of 10% calcium chloride solution. The combination of calcium chloride and thrombin is then mixed with the isolated PRP at a ratio of 1:6 8 to initiate the fibrin gel formation within the graft prior to transfer. It is well known that calcium and thrombin are required for the conversion of plasma fibrinogen, factor VIII and factor VIIIa to fibrin. The exact ratios of mixture are determined by the quality of the formed clot. The physical properties of the formed clot are dependent primarily on the hemodynamic state of the patient, his or her platelet count, and the fibrinogen content. The properties of the clot are further influenced in autologous fat grafting by the difference in volumes and aqueous content of the graft. The desired character is substantially different than when such techniques are applied in orthopedic, maxillofacial, cardiovascular, and neurosurgical applications. 1,3,15 The ratio suggested in this presentation represents a clinically tested sample, and is the subject of ongoing research. We are investigating the possibility of enhancing the quality of the clot while preserving the ability to easily transfer the grafts into the prepared recipient sites. By incrementally adding the calcium chloride and thrombin to the fat graft during gentle, continuous agitation of the preparation, it may be possible to establish the ideal consistency for graft placement. The enriched fat graft is considered ready for transfer after the allotted 10-minute delay accompanied by a congealing effect. When the treated PRP is added to the prepared graft, there is a visible increase of extracellular fluids from the cellular elements. (Figure 5, A and B; Figure 6A) This increase of extracellular fluids leads to an average reduction of the measured fat volume by approximately 60%. This visible concentration of the fat for transplantation is considered of value because it means that the graft contains approximately 60% more cellular components in comparison to conventional methods of handling and transferring autologous fat in a suspension form (Figure 5B, 6B). The condensed graft material may then be placed in various size injection syringes with large-bore cannulae or needles for the actual transfer into the pretunneled recipient bed. Clinical Observations of PRP-Enhanced Fat Grafts and Platelet Gel The addition of PRP and calcified thrombin appears to congeal the graft, establishing a meshwork of fibrin (so-called fibrin gel) that may serve to stabilize the cellular components of the graft and simultaneously provide additional growth factors known to promote healing and ingrowth of capillaries. Also, as in grafting of bone, formation of a carrier gel associated with isolation of PRP and its addition to the graft materials may serve the biochemical intent of introduction of appropriate growth factors while offering enhanced stability of the graft after placement into the pretunneled recipient bed. As in most autologous grafts, relative immobilization of the grafted material in the re-

6 64 The American Journal of Cosmetic Surgery Vol. 18, No. 2, 2001 Figure 5. (Left) The fat aspirate prior to any manipulation. Note the level of fluid, lipid, and cellular debris. The total aspirate volume is 1400 ml. The amount of supernatant fat aspirate can be estimated at about 1000 ml. (Right) The fat aspirate after its purification with normal saline. Excess fluid has been removed. Note the fatcell level remains at just under 1000 ml. cipient site is usually desired during the earliest response phases of healing. Preliminary clinical results suggest improved augmentation and volume retention of autologous fat grafts. In addition, the mixture of PRP with fat appears to reduce the transfer of nonviable extracellular fluid volumes, facilitating more precise augmentation with less requirement to overfill the site to be corrected. The recipient site of PRP-enhanced fat grafts is grossly indistinguishable from the conventional recipient bed. There are, however, some subtle differences in early and late healing stages as the graft becomes incorporated at the site. These may prove of value with reduction in requirement to overfill, and the sites demonstrate an easily palpable increased density in the area of augmentation. Use of gentle massage during the transplantation phase seems effective in site molding and adaptation of graft to the created recipient site tunnels. After transfer of PRP-enhanced grafts, there appears to be less swelling and bruising in the grafted sites. During the second and third postoperative week, recipient sites have remained firmer to palpation than those sites created by use of conventional grafting techniques. This characteristic is transient and appears completely resolved within 4 weeks. Results This technique is currently being investigated on both small and large volume transfers. Initial clinical findings are very encouraging from the standpoint of reduction of loss of fullness associated with the expected gradual removal of extracellular fluids used to transport the grafts into the recipient beds. The apparent concentration and greater quantity of cellular grafts observed after utilization of these techniques seem to yield a higher proportion of graft volume retention. In following the protocol described in this publication, certain consistent changes in the harvested graft elements are noteworthy. As would be anticipated in the formation of a gel-like matrix, the fat concentration yields a firmer consistency. This effect feels similar to the consistency of fat grafts that have been stored in a

7 The American Journal of Cosmetic Surgery Vol. 18, No. 2, Figure 6. (Left) The fat aspirate with platelet rich plasma and calcified thrombin resulting in contracture of the supernatant fat volume and its separation from the excessive fluid. Note the dark spaces within the fat represent excess fluid. (Right) The platelet rich plasma enriched fat. There is a volume reduction of the supernatant fat aspirate by 40 60%. fresh frozen protocol and restored prior to transfer. Because of the denser consistency, we have selected larger bore introduction instrumentation in order to minimize the pressures of delivery into the recipient sites. For large volume transfers, blunt cannulae of mm have proven efficient and effective. In small volume transfers, use of polished titanium, beveled Tulip needles of a minimum of 2.1 mm diameter are used. In order to inject the small aliquots of graft materials through smaller bore devices, cells are exposed to increased pressures that are expected to be harmful to cellular integrity. In addition, we believe that effective but limited pretunneling with polished needles or blunt cannulae will likewise create a small corridor of potential space to receive the graft without forcing it against closed resistance. Risks and Complications Reports of complications associated with autologous fat transplantation are relatively few and infrequent. The most common relate to donor site morbidity and are comparable to type, frequency, and severity in standard liposuction procedures. The recipient site is subject to the influence of the fat graft, any additives, and long-term healing effects common to other graft donor and recipient sites. Palpable nodules after autologous fat grafting have been described. 4,5,8 11,34,35 We believe that palpable nodules after fat transplantation are influenced by delayed healing and should be considered when evaluating these techniques. Similarly, liponecrosis resulting in formation of microcysts and calcification are possible in both small and large volume transfers. 9,34,35 We believe that PRP-enhanced fat grafts may reduce the frequency of development of such findings because of potential acceleration of the revascularization process and graft acceptance. 5 The described technique utilizing fibrin gel requires the addition of thrombin in order to create the stabilized graft. Such thrombin is typically derived from bovine sources and has been implicated in the production of antibodies to bovine thrombin and co-immunization to factor V. 24,25,36 38 The

8 66 The American Journal of Cosmetic Surgery Vol. 18, No. 2, 2001 clinical relevance of this finding in this application has not been determined. Discussion The literature is replete with information regarding a number of biochemical additives and agents believed to influence adipocyte development or differentiation, or both, in vitro and in vivo. Such studies are based on the hypothesis that such agents may be capable of altering the metabolic activities or of inducing development or differentiation of precursor forms. These agents include heparin, calcium, insulin, thyroid hormone, bezafibrate, and vitamin E. 16,17,39 42 Several of these agents were described as being lipogenic or providing inhibition of lipolysis without substantial evidence of changes in the differentiation process. There is little irrefutable evidence that any single additive has produced remarkable effects on the metabolic or developmental actions of the adipocyte population or graft acceptance. Most of the studies remain difficult to compare on the basis of the techniques and research models utilized. For example, in vitro cultures often utilize animal serum that is known to be rich in growth factors ,16,17,40 46 It is possible that some biochemical agents interact by potentiation of various growth factors while individually exerting no direct influence on the adipocytes or potential precursor cell population. However, in several studies, findings of potential importance have been reported. Any biological additive or atmospheric change facilitating improved recipient site vitality would likely contribute to improved consistency and effectiveness. It is believed that viable fat cells do indeed survive the transfer process and, after certain intracellular accommodations, return to active intracellular lipid storage. One recognized limiting factor may be presence of excessive extracellular fluid and cellular debris. Long-term results of fat transplantation have led surgeons to overcorrect the transplanted volume by up to 30 50%. 5,11 Our observations regarding the substantial reduction in graft mass associated with addition of PRP, calcium chloride, and thrombin may make this amount of overcorrection less important. The factors under the surgeon s control that appear to influence the success of fat grafting are most easily divided into distinct steps involved in the grafting process. The steps may be practically broken down into the following categories (Figure 2). Selection of Appropriate Donor Sites Autologous fat appears to display donor-site memory (ie, it may retain the metabolic and storage characteristics of adipocytes common to the sites selected for harvest). This has significant clinical importance in determination of ideal donor site areas, making selection of donor sites not on the basis of surgeon convenience, but on the basis of the demonstrated fat storage capabilities and fat metabolic activity levels in each individual patient. Ideal donor sites are considered those locations in which cells are genetically designated as active in storage. These are metabolically resistant locations (ie, to exercise and diet) commonly referred to as primary deposit sites. These donor areas strongly follow a familial pattern, demonstrating tendencies of certain diet and exercise resistant deposits that appear to correlate with female-to-female or maleto-male phenotypic expression. Selection of Appropriate Recipient Sites In early experimental models, including those by Eppley, 23 investigators often selected recipient sites not naturally native for adipocytes. This may have influenced findings because such areas may have excluded utilization of the potential of a naturally existing source of native precursor cells to act as an additional substrate for the resident or recruited growth factors influencing graft acceptance. Recent animal studies of fat grafting into sites bearing other native fat and connective tissue elements may have demonstrated success when grafts were placed in fat-bearing beds. Resident tissue contributions may produce significant potential benefit by exerting effects on the autologous fat cells being transferred, as well as providing readily available pluripotential cells within the host recipient site. 47 Low Pressure Harvest of Autologous Fat Lipoharvesting appears to have improved high cell viability when grafts are removed by the use of lowpressure closed-syringe techniques. 5,30,31 The harvest phase is directly influenced by volume requirements, location of primary deposits (genetically predisposed deposit sites), and amount of fat available for transplantation. The graft extraction phase is influenced by the thorough distribution and use of adequate tumescent solution into the donor site prior to pretunneling and application of low-pressure vacuum. Provision of an adequate fluid medium (tumescent fluids) is essential in order to facilitate mobilization of fat cells from the investing matrix and to deliver a vehicle to extract the saline cellular suspension. Larger bore, atraumatic cannulae are now favored, with use of closed syringe harvesting techniques for large and small volume removal. Niechajev and Sevcuk 5 reported that suctionassisted aspiration of fat cells under maximum negative pressure (machine suction) did cause cellular damage and vaporization of fat cells. The optimum and least traumatic aspiration pressure was reported to be at or less than 0.5 atm. 5 These findings were confirmed when early reports of the use of a closed-syringe system demonstrated substantial reduction in cellular and site trauma using syringes and low aspiration pressures in a system devoid of air. 31 Reduction in rapid shifts of pressure experienced at the cannula tip is believed to lessen the cavitation and damage of the cells at the time of harvest. Handling and Preparation of Harvested Fat In the belief that solutions that are excessively hypertonic or hypotonic may damage the harvested cells,

9 The American Journal of Cosmetic Surgery Vol. 18, No. 2, the most common medium in which to extract cells continues to be sterile saline or balanced salt solutions. It is believed that thorough rinsing of the harvested graft tissues not only reduces the intracellular lidocaine concentrations, but effectively reduces debris and free lipids from the graft. 32 Rinsed and maximally cellular grafts may result in a less intense inflammatory reaction within the host bed. Exaggerated inflammatory cellular and vascular responses may not be desired during the early healing phases associated with any graft acceptance. During the handling and preparation phase, the opportunity arises to consider additive materials that may influence the adipocytes to be transferred. Identification of potential additives that stimulate cellular metabolism and healing should be considered to enhance autologous fat graft acceptance and retention. Serial rinsing of harvested fat cells may also play an important metabolic role by reduction of intracellular lidocaine levels, which are reported to exert an undesirable metabolic effect. 32 Other authors have suggested the use of Eagle medium to cleanse impurities prior to transplantation. 5 The clinical benefits of such practice is unknown. The importance of reviewing the phase of graft preparation and handling is that it is during this time that it is possible for the surgeon to introduce certain nutrients or other potentially beneficial factors to the graft. Any biological additive or atmospheric change that facilitates improved recipient site acceptance and graft vitality would be contributory to improved efficacy and retention. There are significant similarities between factors that are well established in grafted bone tissues and those that are apparently involved in the grafting of autologous fat cells. It is probable that ongoing research will demonstrate a significant link between mesenchymal-derived stem cells common to both tissues. Animal studies utilizing fibroblast growth factors have suggested significantly increased adipocyte viability and enhanced collagenous ingrowth in the recipient site. 23,48 Transfer Techniques Into Recipient Sites In keeping with the belief of importance of handling cellular grafts with minimal stresses, use of large diameter blunt needles or cannulae ( mm diameter) are thought to be important in minimizing pressure trauma during both extraction and implantation. As in any graft, a healthy recipient site with adequate supportive inflammatory potential, vasculature, and nutrient supply system is critical. 5 The final result of transplantation is thought to be directly dependent on the initiation and maintenance of proper wound healing mechanisms. These then promote early integration and survival of the transplanted fat cells. On removal and transplantation of fat cells, typical ischemic and hypoxic conditions appear, with initial cellular vitality dependent on plasmatic imbibition during revascularization occurring in a centripetal manner. 5,7 It is currently believed that small aliquots of fat should be transferred into prepared tunnels to protect the most centrally located fat cells from potential central necrosis and liquefaction. 4,5,8 11,31 During grafting of fat tissue, care should be taken to avoid large volume clumps of cellular elements in order to reduce this potential. This effect is believed to be a major factor in reports of variable and unpredictable resorption rates. 5 Preliminary findings in use of PRP-enhanced autologous fat grafts suggest that they appear to be advantageous for use as a soft tissue augmentation agent, with the potential of improved acceptance and retention over conventional fat grafting techniques. At this time, the procedure is designed for sequestration of a relatively large volume of blood and currently appears best suited for larger volume transplants that are needed for retroglandular breast enlargement, smoothing trochanteric depressions, posttraumatic defects, idiopathic subcutaneous fat atrophy, or various lipodystrophic conditions. The authors are refining this process to accommodate small volume transfers that would be utilized in lip, cheek, and chin augmentation and in effacing nasolabial folds and smaller scar depressions. Small volume blood sequestration requires a smaller centrifugation bowl to provide properties identical with the larger volume techniques. Such a bowl currently exists, and clinical trials remain in process. Initial clinical findings are very encouraging with utilization of more condensed graft placements. We have observed that the apparent concentration of grafts seem to yield a higher proportion of correction with less volume of graft required. Although these findings are consistent, there is currently no capability for specific quantitation of graft acceptance and survival. In the near future, it is anticipated that we will be able to effectively label and harvest mature adipocytes without interruption of metabolic activities. We will then be able to quantitate the actual numbers of surviving graft cells. To date, efforts to label such grafts have precluded us from being able to perform calculations of surviving and proliferating adipocytes because of interruption with normal fat metabolic activities. Enrichment of grafts utilizing PRP and growth factors in autologous fat transplantation surgery has significant potential to enhance the efficacy of soft tissue augmentation. By such enhancement, it is possible that additional understanding and clinical predictability can be realized. Adhering to strict fundamental biological and surgical principles during the entire adipocyte harvesting and transfer process is believed to represent the most likely keys to success. In other words, fat grafts must be gathered, handled, and transplanted with great care. Abuses during the extraction and preparation are common and may explain the wide diversity of reported experiences and claims. We are currently in the process of working with cell biologists in the process of developing the ability to label the harvested adipocytes without interfering with their vitality and metabolic functions. The natural advantage of such labels would be to quantitate those adipocytes that survive the grafting processes and remain in the augmented

10 68 The American Journal of Cosmetic Surgery Vol. 18, No. 2, 2001 bed. To date, several labels have been added that can be identified and quantitated, in vitro, that may prove useful in this effort. Additional research is needed to establish the same ability in vivo. Consideration must be given regarding use of a platelet gel and incorporation of additives with autologous fat grafts, including the inherent risks and longterm effects of such additives. The described dosages and ratios of PRP, calcium chloride, and thrombin have been modified for fat grafting from a variety of other unrelated clinical applications. 1,3,33 Altering the calcium chloride or thrombin to PRP ratio to 1.5:10 does seem to bind more platelets, reducing their quantities in the liquid media. Potential stimulation of certain antibodies may be directly associated with the amount of bovine thrombin applied and the application. 24 For example, a coagulopathy has been reported in cardiovascular and neurosurgical procedures when CaCl and thrombin were used in large amounts. 24,25,36 38 Similar findings have not been identified with other procedures using similar techniques in smaller volumes. 1,3 In using large amounts, most patients reportedly developed only a subclinical coagulopathy and did not demonstrate any clinical evidence of excessive bleeding. 25 Considering the very small volumes utilized in this protocol, it is extremely unlikely that clinically significant coagulopathy will ever be a factor. Patients undergoing major, large volume uses can be monitored by hemostasis test panels that are activated partial thromboplastin time, prothrombin time, bovine thrombin time, and a factor V level. In the few reported instances, most patients spontaneously reversed their profiles and returned to normal within 3 6 weeks. 25 It is important to develop additional clinical experiences using small volume exposures to establish optimaldose response curves for these compounds and to seek alternative methods of activation of PRP for fat grafting. There are ongoing efforts to develop autologous thrombin from the sequestered blood components. The use of autologous thrombin will alleviate the concern of using a bovine source. This paper presents an effective, simple method of isolation of PRP and platelet gel for the potential use in the application of autologous fat grafting. Growth factors are undisputed in their role in the wound healing process. We suggest that addition of such factors to autologous fat grafts may enhance the survival potential of adipocytes and encourage differentiation of preadipocytes. Long-term clinical documentation and quantitation of such grafts is needed to determine the ideal clinical application of such techniques. Early clinical trials are suggestive of improved proportion of graft volume retention than in the conventional fat grafting protocols we have previously employed. At present, the most common method to objectively quantify the amount of remaining fat after transplantation is by volume differentials and clinical photography. Use of computer surface scanning technology may be valuable in calculating alterations in facial or body tissue augmentation. These clinical methods are operator sensitive, and usually are limited to 1 observer when accumulating data for interpretation. Conclusions Because of the availability of autologous fat cells and the technological means to gently harvest and transfer such cells to tissue needing augmentation, the development of standards to improve predictability, increase augmentation capabilities, and enhance safety and efficacy is very important. As in bone graft technology, concentrated autogenic growth factors offer significant potential for enhancing all of these features. We have discussed the potential advantages of the addition of growth factors to lipoharvested cellular fat grafts relative to their potential favorable influence as chemotactic agents and stimulators of increased numbers of cells competent to initiate and participate in wound-graft healing. Adipocytes have been shown to have specific receptors for such growth factors, with a direct influence on replication and development of mature adipocytes. The integrity and healing potential of the recipient bed is believed to be further enhanced by the angiogenic potential offered by availability of growth factors. PRP may prove to be an ideal additive to autologous fat grafts, and is worthy of considerable laboratory and controlled clinical trials. This concept and theory presents several questions that deserve attention. There is great need for further studies in areas of quantifying survival of transferred fat cells, exploring metabolic considerations to enhance graft survival, and standardization of techniques for study of fat transplantation. Acknowledgment The only tragedy that is more tragic than having an incurable disease is to have a disease for which there is a cure and be unable to find it. Alon P. Winnie In loving memory of Debrah Ann Pacheco who lost her life to breast cancer. Debrah was the procurer of platelet rich plasma in this study. References 1. Whitman DH. 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