gms | German Medical Science

Lipofilling or autologous fat transplantation is increasingly used for various indications in plastic surgery. These include small volume filling prodcedures, and extend to large volume lipotransplantation for breast augmentation or breast reconstruction procedures [1]. For latter procedures retrieval rates of greater than 70% of the inocculum have been reported, but volume loss remains a major sequela of lipofilling procedures. Damaged cells and subsequent necrosis of the injected free fat grafts may account for early volume loss [1], [2], while apoptosis of injected cells may be responsible for long term volume reduction [2], [3].

Improved liposuction techniques and aspirate handling have improved initial viability. However, techniques to improve subsequent in vivo lipoaspirate survival are sparse. An antiapoptotic approach and improved angiogenesis could have a beneficial effect on adipocyte survival [4].

Studies on cytoprotection analysed the effect of EPO in tissues like the brain [5], the kidney [6], myocardial tissue [7], the retina [8], the liver [9] and vascular tissue [10].

Reduction of apoptosis and cytokine induced tissue destruction as well as stimulation of angiogenesis was demonstrated by the application of EPO in vitro and in vivo studies [11]. Anti-apoptotic actions in vascular smooth muscle cells and endothelial cells [12] have been described as well as the prevention of cytochrome c release in mitochondria and suppression of caspase activity [13].

The aim of the present study was to evaluate whether an in vitro single-dose treatment with EPO will reduce apoptosis in an extracorporeal rat organ perfusion model, which we have previously described [14], [15].

In this experiment inguinal flaps (n=5) based on a branch of the femoral vessel, as described in a previous publication were raised in male Lewis rats, weighing between 270 g and 300 g [16], [17]. The technique was performed according to the current regulations and principles of the German law on animal welfare. The animals were housed on a 12-hour light/dark schedule and received water and stock diet ad libitum.

Using an animal narcotic unit (Euthanex, World Precision Instruments, Berlin, Germany), the operative site was shaved and disinfected with Octenisept, an octenidine and phenoxyethanol solution under isoflurane anaesthesia. The abdominal skin was incised in a midline approach and a reversed L-shape extension was performed to the inguinal area bilaterally. The inguinal adipofascial flaps were dissected under an operating microscope (Wild M 691, Heerbrugg, Switzerland) using micro instrumentation. The femoral vessels were proximally ligated. The adipofascial flap was based on the distal femoral artery.

A blunt tipped needle was inserted into the distal femoral artery. The flaps were flushed with 10 cc heparinised saline to wash out residual blood. Next, 1,000 IE EPO in 0.5 cc of 0.9% saline (ERYPO^® FS 1000 I.E./0.5 ml, Jannsen-Cilag GmbH, Neuss, Germany) were injected into the flap via the femoral artery. Afterwards the flap was placed in the bioreactor.

The flaps were continuously perfused in the bioreactor system using Hannover solution as previously described [16], [17]. Briefly, the closed bioreactor system is based on a 500 ml Schott Duran^® bottle, filled with 300 cc Hannover solution [16], [17] attached to a cable pump (Ismatec Reglo digital ISM 834) running with a continuous flow of 2 cc/min. The top had an access and exit for an oxygen permissible pipe, measuring 120 cm in length, and another access for air through a bacterial filter.

Hannover solution has been described as an optimal preservation and culture medium combining the effect of various substances, all of which have been proven to support adipocyte viability [16], [17].

Hannover solution alone without EPO pretreatment from a parallel experiment [17] served as negative control.

All flaps were continuously monitored for 10 days. Tissue specimens for immunohistochemistry were harvested directly after raising the flap before placement in the bioreactor (=0 hrs) and at 8, 24, 48 and 240 hours after permanent perfusion. Specimen were immediately shock-frozen at –80°C.

Immunofluoresence detection was performed on 10 µm frozen sections. Six sections of each flap were used for immunofluorescence histological analysis for cleaved caspase 3 (Cell Signalling, Danvers, Massachusetts, USA, dilution 1:200). Alexa Fluor 546 (donkey anti rabbit IgG, Invitrogen, Eugene, Oregon USA) was used as secondary detection antibody. Counterstaining for nuclei was performed by using Vectashield mounting medium for fluorescence with DAPI (Vector Laboratories Inc., Burlingame, CA, USA).

An antibody against Erythropoeitin receptor (EPO-R) (A364, Novus biologicals, Littleton, Colorado, USA, dilution 1:50) was used to detect EPO-R expression on rat adipose tissue, rat and human endothelial cells from a vessel specimen (positive controls). Negative controls without primary antibody were included. Alexa Fluor 488 (chicken anti rabbit IgG, Invitrogen, Eugene, Oregon USA) was used as secondary labeling antibody.

Fluorescence microscopy was performed using a Zeiss microscope (Zeiss 200M inverted microscope) with Axia Vision software version 4.6.3, Carl Zeiss, Jena, Germany. ImageJ public domain Java image processing software, National Institute of Mental Health, Bethesda, Maryland, USA was used for quantification of apoptotic reactions. One random field of each section was analysed.

Using the SPSS standard software package (version 17; SPSS Inc., Chicago, IL, USA) a statistical analysis was performed, using the Greenhouse–Geisser test of within-subjects effects as a general linear model for repeated measurements and a t-test was used. Results were considered significant if the p-value was <0.05.

Immunofluorescence examination of the fat tissue treated with erythropoeitin revealed that the extent of apoptosis in the adipose tissue was not significantly different from the results of treatment with Hannover solution alone. Data of Hannover solution preserved flaps have been published earlier [17].

Apoptotic reaction was detectable at all time-points increasing from 3.1 ± 2.7% at 0 hrs to 29.4 ± 6.9% at 240 hrs. The difference in apoptotic rates increased significantly (p<0.05) between the consecutive timepoints. These results are shown in Figure 1 [Fig. 1] and Figure 2 [Fig. 2].

Staining for the erythropoetin receptor (EPO-R) revealed an intense positive immunoreactivity for EPO-R on human and rat endothelium (positive controls). However, no immunoreactivity was found in the adipose tissue of the rat at all time points. As expected no immunoreactivity was observed on negative controls (Figure 3 [Fig. 3]).

While fat tissue has many characteristics it makes it a unique source of cells and tissue for the reconstruction of soft tissue defects, however, limitations are given by its restricted tolerance to ischemia [18]. Recently the first objective studies based on MRI volumetry have shown a volume survival rate from 64% to 76 % of injected adipose tissue [1], [19], [20]. Thus, hypoxia and ischemia are considered as possible reasons for adipose tissue absorption after free grafting. In addition apoptosis seems responsible for long term volume reduction in free fat grafts [4]. It was shown that apoptosis of fat tissue can be observed in vitro under serum free conditions. A decreased rate of apoptosis in free fat grafts has been successfully achieved in vivo by adding coenzyme Q10 [21] or EPO [22]. EPO has also been used to stimulate cell growth of various tissues. EPO promotes angiogenesis and increases nutritive capillary density. Cytoprotection is another described effect of EPO. Harder and co-workers [23], [24] administered EPO i.p. in mice on three consecutive days before surgery and evaluated musculocutaneous tissue by intravital microscopy thereafter. They demonstrated that EPO (500 IU EPO/kg body weight) improved capillary perfusion and decreased apoptotic cell death [23].

In a mouse model Hamed et al. demonstrated an antiapoptotic potential of EPO but also found an anti-inflammatory effect of the cytokine. They demonstrated that EPO decreases the rate of fat resorption [22]. The authors discussed either an antiapototic action of EPO as demonstrated by a deceased active caspase 3 activity or the angiogenetic potential of EPO [22].

In contrast to the studies by Hamed et al. we could not find any relevant differences in apoptotic cells in those fat flaps, which were perfused ex vivo receiving a single injection of EPO. The chosen technique ensures the exposure of the whole capillary tree of the fat flap to the administered EPO. We intended to apply a concept that could be transferred to clinical fat transplantation. Therefore, instead of administering EPO systemically [22], [23] we added EPO locally to the tissue. For clinical use we calculated a dosage of 33 IU/kg body weight in an average 60 kg patient with 2,000 IE EPO for 600 cc of transferred fat tissue (analogous to 1,000 IE for 300 cc of Hannover solution in the bioreactor). Thus, side effects should be minimal [23]. High dose EPO therapy might bear the risk of hyperviscosity of the blood, microthromboses, endothelin induced hypertension, enhanced peripheral vascular tonus or even vasoconstriction [25], [26], [27], [28], [29]. Harder and co-workers demonstrated that 5,000 IU/kg body weight EPO injected intraperitoneally, but not 500 IU/kg body weight increases hematocrit [23].

In our presented study we could not detect a reduction of apoptotic cell death with EPO in in vitro fat grafts. One possible reason may be the fact that we could not detect the EPO receptor on the rat fat tissue. Interestingly Hamed and coworkers recently demonstrated a dose dependant antiapoptotic effect of EPO in human fat tissue [24]. We can exclude difficulties in the staining procedure as positive controls showed clear expression. Elliott et al. discussed the problem that some antibodies against the EPO receptors are not specific enough and can also bind to other cytokine receptors [30].

Ercan et al. analysed the anti-apoptotic effect of erythropoietin on apoptosis-induced human mesenchymal stem cells. They treated one group of cultured cells with recombinant human erythropoietin. Apoptosis was analyzed by flow-cytometry and immunohistochemical staining for caspase 3. Cells co-cultured with erythropoietin did significantly show less apoptosis in this experimental setting [31]. These findings are in contrast to our findings.

The correct interpretation of the currently available results and their comparison with other studies should be done with caution and appropriate controls have to be included.

Sabbatini et al. presented another possible mechanism, why EPO administration might work. Lipoaspirates extracted by manual liposuction were seeded in appropriate culture and treated for 3 weeks with 3 doses of EPO [32]. These authors applied CD31 and CD68 immunohistochemistry to identify microvessels and several infiltrating leukocyte cells Their findings demonstrate provascular properties of EPO, as EPO induced an increase of CD31-positive microvessels. CD31 reactivity is regarded to be a marker for activation of microvessels in tissues. Furthermore EPO was able to show a reduction of macrophage number mirroring a reduced inflammatory state. Sabbatini et al. concluded that EPO treatment may be a useful strategy to reduce the inflammatory state of fat transplants and to sustain its revascularization [32].

In this ex vivo perfusion bioreactor model, apoptosis in adipose tissue of rats could not be reduced by treatment of the fat flaps with EPO. Possible reason for this finding is the fact, that the EPO receptor could not be detected in rat adipose tissue, although it was detected in rat vessels. We conclude that previously demonstrated beneficial effects of EPO in vivo are rather due to an improved neoangiogenesis or reduced inflammation than to a direct reduction of apoptosis in the adipose tissue itself via EPO receptor.

The authors declare that they have no conflict of interest concerning the products presented in this paper.