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Autologous Conditioned Serum Increases Fat Graft Viability M… : Plastic and Reconstructive Surgery

The autologous fat grafting for aesthetic purposes is a popular procedure in plastic surgery, representing 5.1 percent of the nonsurgical procedures, with more than 542,000 procedures performed worldwide in 2018 according to the International Society of Aesthetic Plastic Surgery report.1 It is well known that the main problem with fat grafting is the highly variable resorption rates, reported to range from 20 to 90 percent.2 As Frame mentioned, no one really knows why fat graft survival is so unpredictable.3 Carpaneda and Ribeiro reported that the central portion of a fat parcel with a radius larger than 2 mm will not survive because of poor direct diffusion and impaired plasmatic imbibition in the initial 24 to 48 hours after fat grafting.4,5 Thus, several strategies have been used to increase vascularity of the surrounding tissue, decrease the distance, or both to increase the predictability of fat grafting.6–8

In 2018, Liu et al. observed that the neutrophils arrived at the transferred fat tissue as early as 24 hours to form a super early inflammation period, with great production of reactive oxygen species or other inflammatory cytokines, and they reported that excessive neutrophil infiltration can damage the healthy tissues, leading to a poor retention rate.9 The associated inflammation increases the fat necrosis and reduces the final volume achieved.10 Eto et al. noted also that at the early stage of fat transplantation, acute ischemia and inflammation occur because of inadequate blood supply and tissue injury, which leads to tissue edema.11 To increase graft survival rates, there have been many attempts, by eliminating the inflammatory mediators.12 Zhan et al. used local indomethacin as a nonsteroidal anti-inflammatory drug and a cyclooxygenase-2 inhibitor to enhance fat graft retention and reported improved adipose volumes explained by inhibition of immediate inflammatory responses and proinflammatory cytokine-induced apoptosis.13 Tan et al. reported that melatonin enhances fat graft volume retention by down-regulating acute inflammatory cytokines, particularly tumor necrosis factor-α.14 Thus, the inflammation at the receptor level ought to be minimized to increase the viability of fat graft. Current studies, particularly in wound healing models, show that interleukin-1β and tumor necrosis factor-α are the key proinflammatory cytokines of this early inflammatory process.15

Autologous conditioned serum is an autologous blood product obtained by incubation of whole blood taken into a tube containing sterile medical grade glass beads and then centrifugation.16 During the incubation period of whole blood, platelets begin to secrete the preformed granules, whereas mononuclear cells synthesize and secrete interleukin-1 receptor antagonist along with a variety of additional anti-inflammatory mediators (interleukin-4, interleukin-10, and interleukin-13) and growth factors such as epidermal growth factor, vascular endothelial growth factor, hepatocyte growth factor, insulin-like growth factor-1, and platelet-derived growth factor.17–19 Exposure of blood to processed glass beads provides a vigorous and rapid increase in the synthesis of various anti-inflammatory cytokines. Interleukin-1 receptor antagonist concentration has been reported to increase 140-fold after incubation, whereas interleukin-4 and interleukin-10 were slightly induced.20 In another study, it was reported that autologous conditioned serum increased the concentration of fibroblast growth factor-2 by 750 percent compared to basal blood concentration, and increased interleukin-1 receptor antagonist concentration by 600 percent.21

We hypothesized that combining autologous conditioned serum fat graft may increase fat graft survival. If this hypothesis holds true, it can be postulated that marked early proinflammatory changes in fat transplantation affecting graft survival may be conceivable by giving a serum rich in anti-inflammatory cytokines and modulatory cytokines—obtained from the patient’s own blood with simple steps of incubation and centrifugation. It has been proven that platelet-rich plasma enhances fat graft survival before. In this study, our aim is to compare possible additive effects of administration of two biological materials, autologous conditioned serum and platelet-rich plasma, in the survival of fat grafts when combined with fat graft in a rat model.

MATERIALS AND METHODS

All experimental protocols used in this study were conducted according to the international regulations and declarations concerning animal experimentation. The study was approved by the local animal ethics committee (No. 17-0042/482). Thirty-seven adult (120-day-old), male, Sprague-Dawley rats weighing between 200 and 250 g were used. Twenty-seven of them were distributed randomly into three equal groups of nine. The remaining 10 rats (donation group)—which were the same species, sex, age, and weight as the investigated rats—were used to prepare platelet-rich plasma, autologous conditioned serum, and fat grafts (Fig. 1).

Preparation of Fat Grafts

The fat graft was taken from 10 rats of the donation group, which were not included in the study. The fat grafts from bilateral inguinal fat pads (each one has a volume of approximately 1.5 ml) were excised. The adipose tissue obtained from each rat was cut into small pieces less than 1 mm to form an injectable fat graft material.

Preparation of Platelet-Rich Plasma

Five of 10 rats in the donation group were used to obtain cardiac blood for platelet-rich plasma after the fat graft procedure (Fig. 1). Blood of rats was collected into standard tubes containing anticoagulant-citrate-dextrose, solution A, at a ratio of 9:1. To isolate plasma, the tubes were centrifuged at 1630 g relative centrifugal force for 5 minutes in a multipurpose centrifuge (NF 800; NUVE Trading Co., Ankara, Turkey). As expected, after centrifugation, whole blood was separated into three components: red blood cell layer at the bottom (nearly 40 percent of the volume), buffy coat layer in the middle (nearly 10 percent of the volume), and plasma layer at the upper phase (nearly 50 percent of the volume). The buffy coat layer and one-third of the lower layer of the plasma was isolated as platelet-rich plasma (Fig. 2).22–24

Preparation of Autologous Conditioned Serum

The other five of 10 rats in the donation group were used to obtain cardiac blood after the fat grafting procedure (Fig. 1). Blood from rats was collected into 10-ml tubes, 2.5 mm in diameter, and containing approximately 200 small glass spheres made of highly purified special borosilicate material (Sanakin; Scientific BioTech GmbH, Dortmund, Germany). All tubes were incubated at 37°C for 3 hours 15 minutes. After the incubation period, the centrifugation was performed as a single cycle of 5 minutes at 4000 rpm. Then, all of the upper serum layer was taken as autologous conditioned serum.20

Surgical Procedure

Fat grafts taken from donors were first placed in larger syringes by removing the plunger of the syringe to eliminate the dead space in fat grafts. Afterward, 0.7 ml injectable fat grafts were placed into 1 ml-syringes containing 0.2 ml of autologous conditioned serum, phosphate-buffered saline, or platelet-rich plasma by using a 16-gauge angiocatheter to eliminate damage to the fat grafts. Injectable fat grafts (0.7 ml) mixed with autologous conditioned serum (0.2 ml), phosphate-buffered saline (0.2 ml), and platelet-rich plasma (0.2 ml) were transplanted between the muscle and subcutaneous tissue as boluses using 18-gauge needles in each zone: upper third, middle third, and lower third of the dorsum, respectively (Fig. 1). This concentration was chosen according to the previous studies which demonstrated that the adjunction of 0.2 ml platelet-rich plasma to 0.7 ml fat offered the highest proliferation rate.25,26 The same procedure was applied to all rats and then 27 rats were randomly divided into three groups for euthanasia. The euthanasia procedure was planned at postoperative months 1, 3, and 5 to compare the early, middle, and late postoperative periods of the fat grafts.

To assess the volume of the injected fat grafts, all rats underwent computed tomography at the beginning (postoperative day 2) and on day on which the animals were euthanized in each group. Computed tomographic scans can distinguish fat density from all other tissues and was chosen over magnetic resonance imaging because computed tomographic measurements are more exact than those of magnetic resonance imaging.27 Computed tomography recognizes different tissues according to their affinity for x-rays in relation to their intensity expressed in Hounsfield units. Fat was distinguished from skin and bone by Hounsfield units, and a user-defined region of interest was established in coronal and sagittal slices by a single, blinded observer (Fig. 3).28,29 Volumes in each group were then measured; then, the height, width, and length of each recipient area of rats were obtained manually drawing on two-dimensional slices at the maximum (Fig. 3). Three calculations were completed per line and the average determined was taken as the height, length, and width. Then, the graft volume was calculated from the following equation30:

$Volume of ellipsoid=43π12×height12×length 12×width$

Histopathologic Analysis

After the radiologic imaging of each group, the rats were euthanized. The skin samples including the fat graft were completely removed en bloc and fixed in a 10% formalin solution for 24 hours, dehydrated through graded ethanol solutions, cleared in xylene, embedded in paraffin, and sectioned in 4-µm increments.30 All specimens were stained with hematoxylin and eosin reagent and Masson trichrome stain examined under light microscopy. A pathologist blind to the groups performed the examination. The histologic parameters evaluated consisted of investigations on integrity, vascularity, inflammation, and fibrosis. To assess those parameters, the specimens were graded as absent (0), mild (1), moderate (2), or severe (3).30,31

Statistical Analysis

Sample size was calculated to allow at least 80 percent power to detect a 20 percent difference in the presence of a 35 percent standard deviation and positive correlation of 0.50. IBM SPSS Version 20.0 Data Analysis System (IBM Corp., Armonk, N.Y.) was used for data analysis. One-sample Kolmogorov-Smirnov and Levene test was used to check the normality and variances. All the groups were compared using the Kruskal-Wallis H test, whereas the Mann-Whitney U test was used to compare two groups with different scores. Data were presented and used in graphs as medians (interquartile range) for the volume and percentage of the groups. Means ± SD were also used to express the result of volumes and histologic evaluations in tables. Values of p < 0.05 were accepted as significant.

RESULTS

All subjects survived throughout the study. The injected fat grafts in all rats were well-defined subcutaneous masses under the three dorsum regions on all days. The volume measurements of the combined fat grafts placed in the back regions were examined after the surgical procedure (postoperative day 2), and at postoperative months 1, 3, and 5 as early, middle, and late postoperative period (Table 1). At postoperative day 2, no statistical difference was seen between autologous conditioned serum plus fat graft, phosphate-buffered saline plus fat graft, and platelet-rich plasma plus fat graft (p > 0.05). At postoperative months 1, 3, and 5, the differences were statistically different (p < 0.05) (Table 1 and Fig. 4).