Review: Proposed Methods to Improve the Survival of Adipose Tissue in Autologous Fat
Mark J. Landau3
Zoe E. Birnbaum2
Lauren G. Kurtz2
Joel A. Aronowitz MD 1,2,3
Autologous fat grafting (AFG) is not a new technique. It was first described by Neuber in 1893 to fill a depressed facial scar [1]. Used only sparingly for the greater part of the next century, AFG was mostly overlooked in plastic and reconstructive surgery until the early 1980 ‘s. In 1987, as the popularity of AFG surged, the American Society of Plastic and Reconstructive Surgeons (ASPRS) issued a position paper on the use of autologous fat grafting in the breast. At the time, they did not condone the use of autologous fat grafting on the grounds that “much of the injected fat will not survive and the known physiological response to necrosis of this tissue is scarring and calcification. As a result, detection of early breast carcinoma through xenography and mammography will become difficult [2, 3].”
Since 1987, the popularity of fat grafting for a variety of indications has only increased and a significant body of literature demonstrated that artifacts resulting from fat grafting, mainly calcifications and oil cysts, can be readily differentiated from malignancy in early breast cancer screening [4–9]. This is due to improvements in imaging techniques as well as an expanded body of data on the subject. In 2009, the American Society of Plastic Surgeons (ASPS) published a new position paper evaluating the safety and efficacy of autologous fat grafting. The ASPS task force determined that autologous fat grafting was a safe procedure with a relatively low rate of complications. In addition, they stated that artifacts could indeed be identified and distinguished from malignancy in early breast cancer screenings [10].
In recent years AFG has become a widely accepted and utilized technique in the field of plastic surgery due to its application in soft tissue augmentation and its regenerative effects on local tissue such as reversal of hyperpigmentation, softening of hypertrophic scars, increased local vascularity, and improvement of radiated tissue [ diverse applications of fat grafting within the field of plastic surgery include facial rejuvenation, hand rejuvenation, breast reconstruction and volume enhancement, treatment of skin photoaging, correction of contour deformities and improvement of senile and diabetic plantar fat pad atrophy. [14–20]. AFG is useful for both cosmetic and reconstructive indications because there is no scarring at the injection site, no foreign material implanted, a low rate of serious complications, and a typically desirable donor site.
The most troublesome and persistent issue in fat grafting however is the unpredictable degree of volume retention of fat grafts after transplant. Across the literature, studies have reported a wide range of reabsorption rates after transplantation [22]. While some of the variability is no doubt due to inconsistent methods of pre and post operative volume assessment used in various clinical studies reported, a lack of predictable volume retention is clearly a characteristic of autologous fat grafting. A better understanding of the fat graft microenvironment in which engraftment occurs is clearly critical to improvement of clinical outcomes. The current state of knowledge of fat graft failure points toward cellular hypoxia as the major factor adversely affecting the engraftment process and thus long term volume retention. Studies demonstrate that mature adipocytes can tolerate hypoxia for approximately 24 hours at normal core body temperature due to the relatively active metabolic demand of their intracellular cytoplasm. In vivo, fat grafting involves placement of a 1–4 mm diameter adipose tissue fragments, each consisting of thousands of individual cells, into a profoundly ischemic adipose tissue recipient site microenvironment. Ideally the graft fragment is initially nourished by diffusion of oxygen and glucose from the surrounding tissue and quickly revascularized through microvascular inosculation and neovascularization. The clinical success of fat grafting attests to the efficiency of this revascularization process. A significant proportion of injected fat, however, fails to successfully engraft due to irreversible anoxic injury suffered by adipocytes before revascularization can occur. Passive diffusion of oxygen and glucose is not sufficient to sustain adipocytes located centrally in the individual graft tissue fragment, i.e., at a depth of more than about 10 cells from the fragment surface and thus anoxia-induced apoptosis and resorption is the fate of the central core of the graft fragment. Improvement in AFG technique is aimed at reducing the number of mature adipocytes located intermediate between the well-perfused outer cell layers and the anoxic central core of the graft fragment that enter an apoptotic pathway and fail to engraft [23].
Studies have improved understanding of the cellular processes affecting the success of fat grafting and offer the possibility of improving the reliability and predictability of fat grafting. This work for example revealed hypoxia-induced factors expressed by adipocytes which are exposed to ischemic conditions. But the identification of an adipose-specific apoptosis pathway induced by hypoxic conditions and which might be affected by changes in operative technique or medication remains an unrealized research goal. Still, it is possible to identify several factors that contribute to high rates of adipocyte apoptosis and ultimately fat graft resorption. These factors include the threshold parameters tolerated by grafted cells to specific hyponutritional microenvironments, anoxia, and physical trauma which occurs before revascularization. The clinical value of this knowledge is supported by studies that show administration of pro-angiogenic factors such as erythropoietin and vascular endothelial growth factor improve the survival of transplanted fat tissue in mouse models [24, 25]. Another salient factor in successful engraftment is the presence of adipose derived stem cells (ASC’s). These cells are pluripotent mesenchymal stem cells which reside in large numbers in adipose tissue. They are concentrated in the stromal vascular fraction of lipoaspirate. These small stellate shaped cells are identified by surface antigens such as CD134 and their ability to form colonies in vitro. It is estimated that 1–3 million of these small stellate shaped cells typically reside in proximity to small vessels of adipose tissue. Adipose stem cells are known to tolerate the conditions associated with harvest and graft injection more successfully than mature adipocytes, participate in the tissue response to these stresses, and direct adipose tissue regeneration. Importantly, the liposuction fat graft harvest process depletes the number of ASC’s in fat graft material. [54, 55].
Harvest, Handling, and Grafting Technique:
Many groups have attempted to determine the optimal techniques for harvest, processing, and transplantation but there is still no general consensus on the most effective technique. This lack of consensus regarding technique contributes to the wide range of graft retention rates reported across the literature. Part of the problem stems from the fact that AFG actually consists of a multiplicity of individual steps from fat harvest, to graft preparation, to fat injection. Most of the component steps at each stage are likely to substantially affect survival of each small adipose tissue fragment which constitutes the injected graft, thereby affecting the permanent graft volume. Compounding the problem of controlling these multiple variables in clinical studies is the difficulty of studying the major end point, i.e., volume retention. Volumetric measurements, although simpler today with more advanced photographic methods, are notoriously difficult and results are often difficult to compare due to significant differences in both the harvest and recipient site. Nevertheless, there is a significant body of knowledge concerning the factors known to affect graft viability and the engraftment process. These factors fall under graft harvest methods, fat graft processing methods, and grafting technique. They are reviewed below.
Fat Harvest Methods: Fat Processing Methods: Grafting Technique:
No strict recommendation can be made about the best processing technique for AFG based on the literature. Various individual studies have found different methods to be superior depending on the protocols used in each study [29]. However, there are common trends and observations reported across many studies. One observation is that grafts prepared using simple decantation contained the highest number of viable adipocytes, but also the highest number of contaminants. Additionally, grafts prepared using centrifugation at a speed above 400 g have decreased viability of adipocytes.
Platelet Rich Plasma (PRP):
Platelets are a vital part of the immune system response to endothelial injury. Platelets, normally inactive, become activated when they come in contact with damaged endothelial tissue and can also be quickly activated in vitro by contact with glass, freezing cycles, or the addition of calcium or thrombin. Once activated, platelets release stores of growth factors which facilitate tissue repair. Growth factor secretion is most intense in the first hour but continues for about 7 days [31]. The growth factors synthesized by platelets stimulate healing and tissue repair through intercellular mediators and cytokines which stimulate angiogenesis and promote cell proliferation, cell differentiation, and extracellular matrix formation[32–34]. While synthetic forms of these growth factors have been previously studied, they are more readily available and more easily acquired for clinical use in the form of autologous PRP. PRP has been used over the last 30 years to promote bone regeneration, wound healing, tendon and cartilage repair, corneal repair, and skin rejuvenation [35–40]. It should, however, be noted that results across different applications of PRP can sometimes be difficult to compare. This is because since the 1970’s a variety of different methods have been used to prepare PRP, leading to significant variation in the composition and outcomes [41, 42].
In terms of improving fat grafting, PRP has multiple potential beneficial qualities. The growth factor stores in PRP allow cells to resist the hypoxic stress experienced within the first few days after fat transfer and promote proper arrangement of transplanted tissue by facilitating production of the extracellular matrix. The growth factors in PRP also promote angiogenesis which facilitates recovery from the ischemia associated with fat transfer. PRP has been shown to improve fat survival rate as well as promote stem cell proliferation and differentiation in vitro [43, 44].
Recently, additional studies have examined clinical applications of PRP-enhanced fat grafting for wound healing, facial reconstruction, and general aesthetic improvements [48]. Studies have demonstrated that the application of SVF and PRP have similar effectiveness in the treatment of post-traumatic lower extremity ulcers and facial scars [65–66].
In 2012, Gentile et al. [49] published clinical results of a study comparing PRP-enhanced AFG and normal AFG in breast reconstruction. They observed that after 1 year, the PRP-enhanced group (n=50) retained 69% of the initial 3-dimensional volume while the control group (normal AFG, n=50) retained only 39%. In 2013, the same group published another paper expanding on their results with the procedure they termed Platelet-rich Lipotransfer [50]. They observed that when using a PRP concentration of 0.5 mL or 0.4 mL of PRP per mL of fat tissue, 70% of the initial volume was retained at 1 year, compared to only 31% in the control group.
In 2006, the research team led by Kotaro Yoshimura published a paper in which they described a method of supplementing the lipoaspirate used for fat grafting with progenitor cells found in adipose tissue, adipose-derived stem cells (ASC’s). They termed this process cell-assisted lipotransfer (CAL) [53]. The rationale behind this technique is that aspirated adipose tissue (lipoaspirate) is generally poor in progenitor cells, which is a contributing factor to poor survival in vivo. Lipoaspirate is poor in progenitor cells for two main reasons. The first is that ASC’s tend to be located closer to major blood vessels in adipose tissue which are avoided during liposuction and other harvest techniques [54, 55]. The second reason is that a portion of the progenitor cells are contained in the fluid portion of the lipoaspirate, which is discarded before grafting [56]. To combat this problem, Yoshimura and his team suggested harvesting excess lipoaspirate and isolating the progenitor cells contained within, which then can be used to supplement the lipoaspirate to create a progenitor-rich graft.
Studies have been conducted using both the stromal vascular fraction (SVF) and pure, cultured populations of ASC’s. SVF is a heterogeneous population of cells which results from the processing of adipose tissue and is composed mainly of various blood cells, pericytes, macrophages, smooth muscle cells and both adipose-derived and vascular endothelial progenitor cells [64]. The use of a pure population of ASC’s was not shown to be superior to using SVF cells. Using SVF cells is advantageous because they do not require culturing, which can take weeks. Instead, these cells can be isolated and injected in the same surgical procedure. Using cultured cells would require two procedures: one to harvest cells and one for the actual grafting. In the initial paper describing CAL, they reported a 35% increase in retention of grafted tissue volume compared to normal fat grafting in a mouse model. This has led to many projects investigating the therapeutic potential of CAL. In 2013, Kølle et al. [67] conducted a randomized placebo-controlled trial to investigate the effects of ASC enhancement on graft survival in humans. Using cultured ASC’s, Kølle et al. reported significantly higher levels of volume retention compared to controls. The ASC-enhanced group retained 80.9% of the initial volume on average, compared to the control group which only retained 16.3% on average. Another study by Wang et al. published in 2012 reported CAL results from 18 patients [68]. They reported retention of only about 50% of the grafted tissue at 6 months. While there is still absorption of a significant portion of fat after grafting, studies on CAL have reported a positive correlation with ASC enhancement and volume retention compared to normal AFG, but how much the retention is improved is still debated and no conclusive dose vs. effect relationship has been established [69].
The independent advances observed with both PRP and ASC enrichment of fat grafts naturally led to the attempt to combine the two. PRP has been shown to increase the proliferation and differentiation of ASC in vitro [70, 71]. By supplementing ASC enhanced fat grafting (CAL) with PRP, researchers and clinicians hope to boost the regenerative effects of the stem cells, while still getting the benefit afforded by PRP enhancement, in order to achieve a method superior to supplementation with either alone.
In a recent study by Seyhan et al. [72], each of these 3 methods (ASC only, PRP only, and ASC + PRP) were compared in rats. They reported that after 12 weeks the PRP + ASC group had the highest weight and volume of fat grafts while also having the highest number of viable adipocytes and blood vessels. Growth factor levels were also the highest in the PRP + ASC group. While in vitro and animal studies are promising, there is relatively no clinical data available on the combination of the two, most likely because of the lack of adequate clinical data on the use of ASC’s alone. Studies have shown that the injection of ASC combined with PRP accelerated wound closure rates in patients with chronic skin ulcers, but the individual contributions of ASC and PRP to wound closure and their possible synergism has not yet been elucidated. [7 3] There are also studies which examine the combination of PRP and ASC’s in areas outside of fat grafting, such as improvement of knee joint function [74].
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Originally published at https://drjoelaronowitzmd.blogspot.com on July 24, 2023.
