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
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:
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
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.
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).
Changes in Fat Volume by Time at Which Animals Were Euthanized between Groups*
|Time Point||ACS plus FG (μl)||PBS plus FG (μl)||PRP plus FG (μl)||
|Postoperative day 2||>0.05|
|Mean ± SD||1287.5 ± 232.8||1193.7 ± 254.1||1087.8 ± 175.1|
|Postoperative mo 1||<0.05|
|Mean ± SD||677.2 ± 136.2a
||289.1 ± 99.8a,b
||494.9 ± 113.2b
|Postoperative mo 3||<0.001|
|Mean ± SD||558.7 ± 109.6c,d
||262.8 ± 90.6c
||327.2 ± 95.1d
|Postoperative mo 5||<0.05|
|Mean ± SD||576.3 ±158.9e
||265.4 ± 105.4e
||391.1 ± 128.9|
ACS, autologous conditioned serum; FG, fat graft; PBS, phosphate-buffered saline; PRP, platelet-rich plasma; IQR, interquartile range (25th–75th percentiles).
*Lowercase superscript italic letters denote significant differences between fat grafts for every additive. The Mann-Whitney U test was used to compare the data between groups with respect to fat graft volumes. Statistical significance was not reached (p < 0.05) where p values for comparisons are not shown.
The percentage change in the volumes formed based on the initial fat graft volume (0.7 ml), regardless of the fluids given in each group (0.2 ml of autologous conditioned serum, phosphate-buffered saline, or platelet-rich plasma), is detailed in Figure 5. At postoperative day 2, the median volume percentage of the autologous conditioned serum plus fat graft group was 174.6 percent and was the highest (Fig. 5). It was observed as 152.3 percent in the phosphate-buffered saline plus fat graft group and 147.6 percent in the platelet-rich plasma plus fat graft group, but the difference was not statistically significant (p > 0.05). At postoperative month 1, the median volumes were decreased below 100 percent in all groups. The medians and interquartile ranges were 97.3 percent (interquartile range, 77.3 to 119.6 percent), 40.4 percent (interquartile range, 30.9 to 46.9 percent), and 72.1 percent (interquartile range, 53.6 to 84.9 percent) in the autologous conditioned serum plus fat graft, the phosphate-buffered saline plus fat graft, and the platelet-rich plasma plus fat graft groups, respectively (p < 0.05). At postoperative month 3, the median volume of autologous conditioned serum plus fat graft group decreased to 82.3 percent (interquartile range, 70.3 to 88.3 percent); whereas in the platelet-rich plasma plus fat graft group, it decreased to 48.3 percent (interquartile range, 31.4 to 57.9 percent); and in the phosphate-buffered saline plus fat graft group, it decreased to 36.6 percent (interquartile range, 29.4 to 43.1 percent) (p < 0.001). At postoperative month 5, the medians of the autologous conditioned serum plus fat graft and phosphate-buffered saline plus fat graft group were slightly increased to 83.9 percent (interquartile range, 58.3 to 102.4 percent) and 40.3 percent (interquartile range, 20.1 to 50.6 percent), whereas the value in the phosphate-buffered saline plus fat graft was more increased to 56.3 percent (interquartile range, 37.7 to 74.9 percent) (p < 0.05). When the percentages were examined by time, the changes in fat grafts mixed with autologous conditioned serum, phosphate-buffered saline, and platelet-rich plasma were statistically different (p < 0.05, for all) (Fig. 5).
Scores of Integrity
At postoperative months 1, 3, and 5, respectively, the autologous conditioned serum plus fat graft group had higher scores than other groups (p > 0.05, p < 0.01, and p < 0.01, respectively) (Table 2). The integrity had an increase in autologous conditioned serum plus fat graft group by time; however, it decreased at the third month in the platelet-rich plasma plus fat graft group, then increased at the fifth month, but these changes had no statistical significance (Fig. 6). Phosphate-buffered saline plus fat graft had a statistically significant decrease by time (p < 0.05) (Fig. 6).
Changes in Histopathologic Evaluation Scores by the Time at Which Animals Were Euthanized between Groups
|Postoperative Mo 1||Postoperative Mo 3||Postoperative Mo 5|
|ACS plus FG||PBS plus FG||PRP plus FG||
|ACS plus FG||PBS plus FG||PRP plus FG||
|ACS plus FG||PBS plus FG||PRP plus FG||
|Mean ± SD||2.2 ± 1.1||1.9 ± 0.8||1.6 ± 0.7||2.4 ± 0.7b,c
||1.3 ± 0.5b
||1.2 ± 0.4c
||2.5 ± 0.5f,g
||1.0 ± 0.7f
||1.3 ± 0.7g
|Mean ± SD||1.2 ± 0.4||1.2 ± 0.4||1.3 ± 0.5||1.3 ± 0.5||1.4 ± 0.5||1.0 ± 0.0||1.2 ± 0.4||1.0 ± 0.0||1.0 ± 0.0|
|Mean ± SD||1.6 ± 0.7||1.6 ± 0.5||1.8 ± 0.5||0.4 ± 0.5d,e
||1.1 ± 0.6d
||1.2 ± 0.4e
||0.3 ± 0.5h,i
||1.0 ± 0.5h
||1.0 ± 0.0i
|Mean ± SD||1.2 ± 0.4a
||1.6 ± 0.5||1.9 ± 0.4a
||1.1 ± 0.3||1.0 ± 0.0||1.1 ± 0.3||0.6 ± 0.5j,k
||1.1 ± 0.3j
||1.0 ± 0.0k
ACS, autologous conditioned serum; FG, fat graft; PBS, phosphate-buffered saline; PRP, platelet-rich plasma; IQR, interquartile range (25th–75th percentiles).
*Lower-case superscript italic letters denote significant differences between fat grafts for every additive. The Mann-Whitney U test was used to compare the data between groups with respect to the histopathologic evaluation scores. Statistical significance (p < 0.05) was not reached where data had no lowercase letters.
Scores of Vascularity
Although the scores for vascularity in all three groups were almost similar ar postoperative month 1, it was observed that the score for vascularity in the autologous conditioned serum plus fat graft group was higher at postoperative month 5 compared to the other groups. However, in all periods, there was no statistically significant difference for the scores for vascularity (p > 0.05) (Fig. 7).
Scores of Inflammation
No statistically significant difference was seen between all three groups at postoperative month 1 (p > 0.05). When the third and fifth postoperative months were evaluated, it was seen that the autologous conditioned serum plus fat graft group had a significantly lower inflammation score compared with the other two groups (p < 0.05, for both) (Table 2). When the change by time was examined, it was seen that the platelet-rich plasma plus fat graft and autologous conditioned serum plus fat graft groups were statistically significant (p < 0.05, for both) (Fig. 8).
Scores of Fibrosis
It was seen that the autologous conditioned serum plus fat graft group had the lowest scores at all postoperative months; however, statistically significant differences were observed in postoperative months 1 and 5 (p < 0.05, for both) (Table 2). The fibrosis decreased gradually over time in the autologous conditioned serum plus fat graft group and the platelet-rich plasma plus fat graft group; however, the score in the phosphate-buffered saline plus fat graft group had an increase from postoperative months 3 to 5 (p < 0.05) (Fig. 9).
Histopathologic Analysis of Fat Grafts
Histologic analysis of fat grafts mixed with autologous conditioned serum showed overall less inflammation compared to platelet-rich plasma and phosphate-buffered saline (Fig. 10, above). When the postoperative month 3 and 5 samples were also examined, it was noteworthy that the inflammation was minimized, the collagen bundles were arranged more regular, and fibrosis was seen less in the autologous conditioned serum plus fat graft group by time (Fig. 10, below).
In our study, the median percentage of volume on the second day was observed to be higher in the autologous conditioned serum plus fat graft group than in the phosphate-buffered saline plus fat graft and platelet-rich plasma plus fat graft groups (174.6 percent, 152.3 percent, and 147.6 percent, respectively). The main reason for such a high percentage in the early period is the volume effect of autologous conditioned serum, platelet-rich plasma, or phosphate-buffered saline, which is given at a ratio of 1:3 in fat grafts. The reason for the difference between the groups may be related to the degree of inflammation. This change was also seen in the study by Nishimura et al. aimed to determine how fat grafts get their vascular supply from the recipient bed and why they keep diminishing in volume and weight.32 They noted that the weight of the graft increased during the first 7 days, then decreased gradually (increased to nearly 138 percent on day 2 and 134 percent on day 7 according to the graphic).32 As known, fat grafts are believed to obtain nourishment from interstitial fluid for the first 4 days after transplantation.32 It is the supportive part of our hypothesis that increased degree of inflammation prevents fluid intake by plasmatic imbibition, which may lead to less volume increase in early postoperative period (i.e., the more inflammation, the less plasmatic imbibition). At postoperative month 1, the volume percentage of phosphate-buffered saline plus fat graft decreased to nearly 40 percent and remained roughly stable in the other months. In contrast, it was seen that the autologous conditioned serum plus fat graft group had the highest value among the three groups, with a median of 97 percent at postoperative month 1, 82 percent at postoperative month 3, and 84 percent at postoperative month 5. However, when the platelet-rich plasma plus fat graft group is examined, it can be seen that it had a median of 72 percent at postoperative month 1, whereas it decreased to an average of 48 percent at postoperative month 3, and then increased again to a median of 56 percent at postoperative month 5. The reason for such difference in the platelet-rich plasma plus fat graft group may be because platelet-rich plasma contains many proinflammatory and preangiogenic factors as a mixture. Although the inflammation that occurs in the first days decreases the nutrition somewhat, during the angiogenesis that occurs after postoperative day 4, new vessels start to be formed, and it may maintain the viability of the fat graft more accurately. This may explain why the volume of the platelet-rich plasma plus fat graft group in the first month is better than the volume of phosphate-buffered saline plus fat graft group. After that, however, inflammation shows its negative effect in the midterm. Nishimura et al. in their study showed that the number of grafted fat cells decreased after revascularization had been established.32 They explained the early volume loss of the fat graft as acute necrosis, whereas they hold the apoptosis responsible for the volume loss in the later period.32 They most often observed apoptotic cells on day 30. After 30 days, they noted that dead fat cells and lipid drops were removed by macrophages, which could explain the continued weight loss of the grafted fat in the later period.32 The leukocytes may have caused inflammation, leading to the excretion of cytokines and thus the induction of apoptosis, which may be responsible for the volume loss after the first month in the platelet-rich plasma plus fat graft group because of more inflammatory cytokines rather than autologous conditioned serum plus platelet-rich plasma group.22 The late response of the increased graft survival rate in the platelet-rich plasma plus fat graft group may result from the principal effect of platelet-rich plasma on the induction of angiogenesis favoring cell proliferation, possibly by stimulating adipose tissue–derived stem cell proliferation and capillary formation.24
When histopathologic scoring values for inflammation are examined, it could be commented that inflammation decreases over time in all groups, but a higher rate of decrease was observed in the autologous conditioned serum plus fat graft group than in the other two groups. Similarly, the scoring values of fibrosis, which is a result of an inflammatory process, was less in the autologous conditioned serum plus fat graft group compared to the other two groups.
Autologous conditioned serum consists of increased concentrations of antiinflammatory cytokines and has decreased concentrations of proinflammatory cytokines. Autologous conditioned serum should not be seen as just an interleukin-1 receptor antagonist, as it has a disease-modifying effect.17 In addition, platelet-rich plasma and autologous conditioned serum also includes a milieu of anabolic growth factors.19 The rich biological contents of both autologous conditioned serum and platelet-rich plasma clarify why those autologous solutions have the capability to increase the outcomes of the fat grafting procedure. Compared to platelet-rich plasma, the antiinflammatory nature of autologous conditioned serum may take autologous conditioned serum one step further as the better additive material in fat graft applications. To our knowledge, no study comparing the effects of autologous conditioned serum and platelet-rich plasma in fat graft survival has been performed before. However, this study is a pilot study aimed to evaluate the effects of two biomaterials on fat grafting. It has some limitations involving a limited number of groups and small volume of fat grafts because of the animals used. It could be expanded by using larger animals with larger volumes in future studies. Moreover, we had not studied possible dosages of autologous conditioned serum on the fat graft viability and further immunostaining methods to reflect the pathways under autologous conditioned serum that might lead to a limited proof. Thus, further experimental studies are undoubtedly needed to determine whether autologous conditioned serum has any clinical significance.
Although both autologous conditioned serum and platelet-rich plasma improved the outcomes of fat grafts compared to saline, our study revealed that autologous conditioned serum was associated with less inflammation, greater fat viability, and more integrity, particularly in the early and mid-terms compared to platelet-rich plasma. However, it is obvious that more preclinical studies are needed to determine the effectiveness and mechanism of autologous conditioned serum on fat grafts. We believe that our study will be encouraging to apply autologous conditioned serum–containing fat grafting procedures on further scientific studies and may lead to other emerging therapies investigating anti-inflammatory and modulatory effects of autologous conditioned serum.
The authors would like to thank Munevver Bilgic for drawing the illustration.