Biological Roles and Prospects of Using Adipose Tissue Derivatives and Platelet-Rich Plasma in Surgical Practice: A Review

2.1. Adipose Tissue and its Importance in Medicine

3.1. History and Modern Approaches

Lipofilling, the transplantation of a patient's own adipose tissue harvested via liposuction, has evolved significantly since its inception. Its history is divided into three periods: (1) 1889–1977, when fat was obtained through surgical excision; (2) 1977–1994, marked by the use of traumatic or unrefined liposuction; and (3) from 1994 to the present, characterized by the refined Coleman method [21]. The first documented use of fat as a filler dates to 1889, when Meulen V. utilized omentum tissue to treat a diaphragmatic hernia [22]. In 1893, Neuber G. performed the first fat graft, transferring adipose tissue from the arm to the orbital region. He noted that smaller grafts yielded better aesthetic outcomes than larger ones [23]. In 1895, Czerny V. presented a case of breast reconstruction using a lipoma to fill a defect after partial mastectomy, a technique later adopted for facial and breast augmentation [24].

In 1910, Lexer E. pioneered the use of autologous fat in cosmetic surgery, applying it to enhance cheekbones, fill wrinkles, and treat facial deformities in patients with Parry-Romberg syndrome. He also used fat grafting for tendon repair in patients with Dupuytren's disease [25]. Brunning, in 1911, introduced fat injection for rhinoplasty, while Holländer E. experimented with combining human and sheep fat to reduce resorption, although his method had limited adoption [26]. The mid-20^th century witnessed further advancements. In 1941, Billings E. and May J. reported breast reconstruction using autologous fat, suggesting that fascia could improve graft survival [27]. During World War II, fat grafting was even used to alter facial features for espionage purposes [21]. In the 1950s, Peer L. revealed that about 50% of grafted fat cells die, with the remaining cells surviving intact [28].

The development of liposuction in the 1970s revolutionized fat harvesting. Arpad and Fischer introduced a surgical technique in 1974, which Illouz Y. refined by using blunt cannulas to minimize tissue damage [29]. In 1986, Ellenbogen demonstrated successful fat grafting for facial corrections, and Bircoll M. reported breast symmetrization using lipoaspirate, although concerns about fat necrosis and cancer detection temporarily halted research [30]. The 1990s brought further innovation. Hang-Fu L. introduced fat-filled implants to avoid complications of direct fat injection [31], while Chajchir A. emphasized careful fat handling, saline rinsing, and grafting into vascularized beds for optimal results [32]. Coleman S. revolutionized the field with his refined technique, focusing on gentle harvesting, cleaning, and precise placement of small fat aliquots to ensure graft survival and predictable outcomes [33].

Currently, lipofilling is widely used to address volume loss due to aging, injury, or congenital defects. Its applications have expanded to include scar treatment, wound healing, and skin rejuvenation. Despite its long history, lipofilling remains a dynamic area of research in plastic and reconstructive surgery.

3.2. Main Stages and Theories of Fat Graft Survival Rate

There is no universally accepted method for harvesting and preparing adipose tissue for grafting despite extensive research on the topic [34]. The lipofilling process typically involves three stages: (1) harvesting fat via small- or large-volume liposuction, (2) processing the harvested fat, and (3) injecting it into the recipient site. The Coleman method, introduced in the 1990s, remains the most widely used technique. It emphasizes minimizing damage to adipose tissue by using low manual negative pressure for harvesting, followed by centrifugation to separate the fat into three layers: oil, cell fraction, and debris. The cell fraction, containing adipocytes and stromal vascular cells, is then injected into the recipient site in small aliquots to promote neovascularization [35].

Since Coleman's method was introduced, various modifications have been developed. Some techniques eliminate centrifugation, opting for rinsing and decanting with Ringer's lactate solution or using sterile towels to absorb liquids and debris [36]. Commercial systems have also emerged, aiming to improve graft viability by maximizing live cell proportions and minimizing cell damage [37]. A consensus study by Kaufman M [38]. found no significant differences in outcomes based on donor sites or purification methods, although most surgeons overcorrect to account for fat resorption, estimating at least 50% resorption after six months.

The success of fat grafting depends on several factors, including cell viability, the type of cells injected (adipocytes, preadipocytes, or stem cells), and the exclusion of harmful factors like residual blood products, which can promote bacterial growth and cell damage [39]. However, some researchers argue that most grafted cells die, and graft survival relies on stimulating local cell proliferation [40]. The role of specific cell types in graft viability remains unclear, and no definitive comparative studies have identified the most reliable technique.

The choice of cannula diameter for liposuction may also impact graft success, although this area is underexplored. Additionally, inconsistent results in the literature may stem from variations in technique, patient factors, or a limited understanding of adipose tissue biology, particularly the effects of aging. The standard Coleman technique involves harvesting fat from donor sites like the abdomen or hips, followed by centrifugation to separate the lipoaspirate from the oil and infranatant layers. The lipoaspirate contains mature adipocytes and the stromal vascular fraction (SVF), which can be further isolated for laboratory use [41]. A major challenge in fat grafting is the unstable survival rate of the graft, often due to inadequate vascularization. Mature adipocytes are highly sensitive to ischemia and may die without prompt revascularization. However, they can recover if the blood supply is restored quickly [42]. Prolonged graft survival could enhance healing outcomes by sustaining the release of healing mediators and supporting stem cell differentiation.

4.1. PRP: Concept, Classification, and Methods of Production

The survival and remodeling of fat grafts are closely associated with the extracellular matrix, which regulates cell migration, proliferation, and differentiation. Several theories attempt to explain how fat grafts survive after avascular implantation. The Graft Survival Theory, proposed by Peer et al. [43], suggests that fat grafts initially rely on plasma diffusion for nutrients until neovascularization occurs, with smaller grafts showing better survival due to improved diffusion. The Graft Replacement Theory posits that most donor adipocytes die and are replaced by new adipocytes generated from donor stromal vascular fraction (SVF) cells, which drive adipogenesis and angiogenesis. Enriching grafts with SVF has been shown to improve retention [44]. In contrast, the Host Cell Replacement Theory argues that grafted cells necrotize and are entirely replaced by recipient cells, forming fibrous tissue, new fat cells, and blood vessels. The integrity and environment of the recipient site play a critical role in this process [45].

Eto et al. (2012) further elaborated on graft survival by dividing it into three zones [46]. The outermost survival zone, up to 300 microns thick, contains viable adipocytes and adipose-derived stem cells (ASCs). Below this lies the regenerative zone, 600–1200 microns thick, where adipocytes undergo necrosis and regeneration. The innermost central necrotic zone is characterized by hypoxia-induced cell death, leading to resorption or fibrosis. Neovascularization begins around 48 hours post-grafting, progressing from the periphery. Without adequate blood supply, most adipocytes in the regenerative and necrotic zones die within 24 hours, releasing inflammatory factors [47]. Revascularization improves within three days, with adipogenesis replacing dead adipocytes over three months. The chronic stabilizing phase lasts up to nine months, during which lipid absorption continues, potentially leading to resorption, fibrosis, or oil cyst formation [48].

Long-term graft retention varies significantly, with some studies reporting up to 80% volume loss [49]. Factors influencing retention include technical aspects like fat preparation and injection techniques [50], patient-specific factors, such as age, BMI, and diabetes [51], and the degree of neovascularization. Inadequate vascularization is a critical limitation, as mechanical pressure during injection can damage fat tissue, reducing graft survival [52]. Adipogenesis requires early angiogenesis, as delayed blood supply leads to adipocyte necrosis [53]. Experimental evidence shows that improved vascularization correlates with higher graft retention [54].

The success of fat grafting also depends on the preparation of the recipient site. Various methods have been explored to optimize this process. Physical impact, such as cyclic negative pressure, has been shown to improve graft survival [55]. Alloplastic material implantation slows fat resorption by inducing a chronic inflammatory response [56]. Vascular endothelial growth factor (VEGF) injection enhances vascularization and reduces oil cyst formation [57]. Ischemic preconditioning improves graft viability by enhancing tissue oxygenation [58], while microneedling stimulates collagen formation and vascularization through mechanical microtraumatization [59]. Preclinical studies suggest that PRP injection into the recipient site before fat grafting can improve graft survival [60], though comprehensive comparisons with other methods are lacking. Further research is needed to refine these techniques and optimize recipient site preparation for better outcomes.

4.2. Combined Use of PRP and Lipofilling

Platelet-rich plasma (PRP) is defined as plasma with a platelet concentration several times higher than in peripheral blood [61, 62]. The term lacks a single consensus definition, with variations, such as platelet concentrates, autologous growth factors, and platelet-rich fibrin matrix [63]. The composition of PRP varies based on preparation protocols, affecting platelet and leukocyte counts, as well as growth factor content [64]. Marx R.E. reported a platelet concentration 4-5 times higher than baseline [61], while Cho J.M. suggested 3-7 times higher concentrations [65]. Over 17 commercial protocols exist, each yielding different PRP compositions [66]. PRP is now defined by an absolute platelet concentration exceeding 1 × 10⁶/µl, a fivefold increase from baseline [67]. Fadadu et al. [68] reviewed PRP systems and observed that some produce platelet counts lower than whole blood. PRP research began in hematology in the 1970s, focusing on hemostasis and fibrin use [69]. Kohler and Lipton discovered platelet-induced fibroblast growth in 1974 [70], and PRP was first used clinically in 1987 during open-heart surgery to reduce blood transfusions [71]. By the 1990s, PRP was applied in maxillofacial surgery [72], leveraging its fibrin adhesion and anti-inflammatory properties [73]. PRP is now used in sports medicine, dermatology, cardiac surgery, plastic surgery, orthopedics, etc [74-79].

In gynecology, PRP aids wound healing post-cesarean section, reducing redness and swelling [80] and alleviating vulvar dystrophy symptoms [81]. In reconstructive surgery, PRP accelerates healing in chronic ulcers [82], although its efficacy in scar treatment and breast reconstruction remains inconclusive [83]. PRP has shown mixed results in burn surgery, with some studies reporting improved graft survival [84], while others found no significant benefits [85]. In dermatology, PRP effectively treats alopecia [86].

The use of platelet-rich plasma (PRP) in lipofilling is based on the premise that PRP's proangiogenic and anti-inflammatory properties enhance the outcomes of fat grafts [87]. Rigotti G [88]. reported that incorporating PRP into lipografts resulted in more pronounced inflammatory infiltrates, increased vascular reactivity and permeability, as well as some activation of neural pathways. However, it did not significantly enhance regenerative effects. Studies by Cervelli V [89], Segreto F [90], and Smith O [91]. evaluated the combination of PRP and fat grafts for wound healing, confirming the method's safety and feasibility. Additionally, it is suggested that the growth factors released by platelets promote the proliferation and differentiation of adipose-derived stem cells, potentially improving grafting outcomes [87]. Segreto F. reported a decrease in pain and a speed of complete healing by more than 50% [90]. According to Cervelli V., lipofilling in combination with PRP accelerates the process of re-epithelialization of ulcers compared to wounds treated with hyaluronic acid and collagen [89]. Smith O., with co-authors [91], conducted a single-center randomized controlled trial (RCT) with parallel groups (control group, lipofilling, and lipofilling with PRP) for patients with chronic non-healing diabetic foot ulcers. They examined 334 patients; 32 patients (9.6%) were found suitable, and 18 patients were included in the study (6 in each group) for 17 months. No significant differences in clinical outcomes were observed between the groups in most studies. However, researchers concluded that the procedure is safe and recommended conducting larger randomized controlled trials to further assess the efficacy of combining lipofilling with PRP for wound treatment. Fontdevila J. investigated the combination of lipofilling and PRP in patients with facial atrophy caused by HIV (human immunodeficiency virus). Participants were randomized into two groups: one received autologous fat injections, and the other received autologous fat combined with PRP. Structural changes in the facial soft tissues were evaluated using computed tomography and photographic documentation at 2 and 12 months post-treatment. The study found no significant differences in outcomes between PRP-enhanced fat grafting and fat grafting alone [92]. Willemsen J [93], who applied lipofilling for gluteal augmentation, noted a shorter recovery period in the PRP group, likely due to PRP's effects on fibroblast growth and differentiation. Similarly, Bilkay U [94]. found that combining PRP with lipofilling reduced the number of sessions needed to achieve satisfactory results. However, this benefit was not observed in breast reconstructive surgery, as reported by Salgarello M [95]. Clinical outcomes, ultrasound findings of liponecrosis, and the need for additional fat grafting indicated that fat grafting combined with 10% PRP did not outperform fat grafting alone. In scar treatment, Tenna S [96]. and Majani U [97]. observed improved aesthetic outcomes and skin quality with PRP. Comparisons between PRP and the stromal vascular fraction (SVF) as adjuncts to adipose tissue in lipofilling showed mixed results. Van Dongen J [19]. and Sasaki G [98]. reported comparable effectiveness between PRP and SVF in facial lipofilling. Gentile P [99, 100], in two studies, demonstrated higher graft survival rates in breast reconstruction and scar treatment when using both PRP-enhanced lipofilling and SVF, with PRP producing superior results in both cases. These findings confirm the potential of PRP as an effective adjuvant in lipofilling and highlight its ability to enhance outcomes in various applications.

Numerous clinical studies have demonstrated the positive impact of PRP on the outcomes of adipose tissue contouring procedures [87, 101, 102, 103, 100], skin rejuvenation [104], chronic connective tissue diseases [105], and wound healing [89, 106, 107]. Specifically, there is scant evidence supporting a synergistic regenerative relationship, as most studies focus on enhancing the survival and preservation of fat grafts. It has been proposed that the fibrin component of PRP may serve as a scaffold for adipocytes, helping to retain them at the graft site for a longer duration [108, 109]. The fibrin lattice can also reduce apoptosis in differentiated adipocytes [110]. Siegel K. reported that stem cells remaining in the fibrin clot demonstrate consistently higher VEGF secretion, as well as increased immunoreactivity, which indicates a synergistic relationship between the fibrin clot and stem cells [111]. PRP also includes cell adhesion molecules, such as fibronectin and vitronectin, which aid in immobilizing growth factors within fibrin and enable the fibrin lattice to function as a matrix for epithelial migration [109]. When adipose tissue stem cells are cultured in vitro on a lattice enriched with adhesion molecules and growth factors present in PRP, they exhibit enhanced differentiation into keratinocytes, highlighting their increased potential for wound healing [112]. In vitro studies have also demonstrated that most platelets remain active for up to 10 days when co-cultured with stem cells, suggesting a mutual survival relationship that could enhance healing potential [113]. PRP possesses anti-inflammatory properties that help reduce inflammation and swelling, which are factors that can contribute to the degeneration of fat grafts [114]. The elevated levels of hepatocyte growth factor and tumor necrosis factor in PRP can exert a significant anti-inflammatory effect by inhibiting pro-inflammatory factors, thereby enhancing the survival of co-cultured cells [115]. Adipose tissue stem cells co-cultured with PRP secrete low concentrations of chemokines [116], and PRP also contributes to the suppression of IL1B [117]. Adipose tissue stem cells cultured with dolphin PRP also demonstrate an increased ability to phagocytosis, which indicates a role in the stage of inflammation during healing [118].

Several in vitro studies have demonstrated that PRP enhances the proliferation of adipose-derived stem cells (ASCs) [119-122] and can serve as a safe and reliable alternative to standard culture media [123, 124]. This increased proliferative effect does not compromise the ability of ASCs to differentiate [125]. A greater volume of ASCs within fat grafts has been shown to positively influence graft retention and survival [126].

Recent studies have highlighted the importance of donor site selection and the thickness of adipose tissue for optimal lipofilling outcomes. Adipose tissue from the abdomen and thighs is often preferred due to its higher concentration of adipose-derived stem cells (ASCs) and better graft survival rates [127]. The thickness of the adipose layer also plays a crucial role, with thicker layers (≥2 cm) providing more viable adipocytes and stromal vascular fraction (SVF) cells, which are essential for graft survival and neovascularization [128]. Additionally, the abdominal region, particularly the lower abdomen, has been shown to yield higher volumes of adipose tissue with superior regenerative properties compared to other donor sites [129]. These findings suggest that careful selection of donor sites based on adipose thickness and cellular composition can significantly improve the outcomes of lipofilling procedures.

One study revealed that PRP reduces preadipocyte apoptosis by suppressing cell-death mediator mRNA proteins and inhibiting pro-apoptotic genes, thereby improving fat survival after grafting [130]. Li et al. observed that PRP upregulates the expression of adipogenic genes in mice, indicating a favorable impact on fat growth. However, the optimal PRP concentration for ASC growth remains unclear, with recommendations varying between 5% and 15% in different studies [131, 132]. High PRP concentrations (40–50%) have been found to induce cell death due to the negative regulatory effects of platelets on growth factors [133].

While PRP enhances proliferation, it alone is insufficient to significantly increase adipogenesis [134]. Chignon-Sicard B. noted that concentrations of 20% PRP could inhibit adipogenic differentiation [135]. Despite this, ASCs retain the potential to differentiate into endothelial cells, a critical step in angiogenesis as they form capillary structures [136, 137].

In vitro studies indicate that co-culturing PRP with ASCs promotes the formation of vascular networks [138], and animal studies suggest that combining PRP with fat grafting enhances neovascularization [139]. PRP also stimulates the differentiation of stem cells into fibroblasts and keratinocytes, which are essential for wound healing [140]. Moreover, it facilitates fibroblast migration to wound sites [141], further aiding the healing process.

ASCs cultured with PRP express matrix metalloproteinases 1 (MMP1) and matrix metalloproteinases 2 (MMP2) genes, which are key players in tissue remodeling, suggesting a beneficial role in wound healing [117]. These findings collectively highlight the potential of PRP to improve fat grafting outcomes and promote regenerative processes, although the optimal conditions for its use require further research. The fibrin component of PRP serves as a scaffold for adipocytes, while its anti-inflammatory properties help mitigate inflammation and swelling, which can otherwise contribute to fat graft degeneration. Furthermore, stem cells derived from adipose tissue possess the potential to differentiate into endothelial cells, promoting angiogenesis. Two studies [142, 143] demonstrated that adding PRP improved graft organization, with histological images revealing a more uniform cell distribution compared to controls. Although these findings were visually evident, quantification and statistical analysis were lacking. This suggests that the fibrin component may enhance graft survival by providing structural support. An alternative hypothesis is that the higher density of surviving cells due to PRP gives the appearance of organization. PRP has been shown to enhance angiogenesis, increasing vascular density by 60–260% within 7 to 14 days [144-147]. Additionally, it boosts the expression of angiogenic growth factors stored in platelet α-granules and increases the thickness of granulation tissue [146]. Preserving viable tissue in fat grafts is critical for wound healing, as non-viable tissue can hinder the process. Studies examining fat grafts without PRP [148] indicate that adipocytes and adipose-derived stem cells suffer initial damage during collection and injection. By day 1, immunological cells infiltrate the grafts, clearing cellular debris, while some adipocytes undergo necrosis. Neoangiogenesis begins at the periphery by day 4, and by day 8, vacuoles and oil-fat cysts (markers of ischemia) are no longer evident in central adipocytes. In mouse models, adipocytes located more than 300 microns from the graft periphery became nonviable within 1 day, but viable cells began reappearing after day 7, with markers like Ki67 indicating cell proliferation [46].

Six studies reported changes in fat graft volume and survival with PRP addition [143, 149-152]. Four of these found that PRP improved adipocyte viability [149-152]. Rapid revascularization is essential for the survival of adipocytes and stem cells in grafts. While most animal studies focused on long-term cell viability (3 to 6 months), only one study evaluated histology at 10 days but did not report results on adipocyte viability at that time [142]. Nonetheless, it is reasonable to infer that initial revascularization determines long-term viability, as nonvascularized cells become hypoxic and die after 7 days. Two additional studies identified indirect indicators of improved viability, such as fewer vacuoles in PRP-treated grafts [143, 150], while one study found no significant difference [153]. Activation of PRP, usually with calcium chloride, has been shown to enhance cell viability more effectively than inactivated PRP. For instance, Hersant B [142]. reported that activated PRP increased graft viability to 24%, compared to 13% and 14% for saline and inactivated PRP, respectively. PRP-treated grafts also showed a 91–97% increase in vascular density [149, 150]. The precise contribution of PRP versus adipose-derived stem cells to increased vascular density remains unclear. Additionally, it is uncertain whether these changes occur quickly enough to prevent early ischemia in grafts. While PRP-induced angiogenesis might lead to greater vascularization later, it could coincide with reduced viability of graft cells if critical revascularization is delayed.