gms | German Medical Science

GMS German Plastic, Reconstructive and Aesthetic Surgery – Burn and Hand Surgery

Deutsche Gesellschaft der Plastischen, Rekonstruktiven und Ästhetischen Chirurgen (DGPRÄC)
Deutsche Gesellschaft für Verbrennungsmedizin (DGV)

ISSN 2193-7052

Autologous fat transplantation – animal models and methods for in vitro analysis of viability

Autologe Fetttransplantation – Tierversuche und Methoden für In-vitro-Vitalitätsanalysen

Review Article

  • corresponding author C. Herold - Department of Plastic, Hand, and Reconstructive Surgery, Hanover Medical School, Hanover, Germany
  • M. Pflaum - Department of Heart, Thoracic, Transplantation and Vascular Surgery, Hanover Medical School, Hanover, Germany
  • P. Utz - Department of Plastic, Hand, and Reconstructive Surgery, Hanover Medical School, Hanover, Germany
  • M. Wilhelmi - Department of Heart, Thoracic, Transplantation and Vascular Surgery, Hanover Medical School, Hanover, Germany
  • P. M. Vogt - Department of Plastic, Hand, and Reconstructive Surgery, Hanover Medical School, Hanover, Germany
  • H. O. Rennekampff - Department of Plastic, Hand, and Reconstructive Surgery, Hanover Medical School, Hanover, Germany

GMS Ger Plast Reconstr Aesthet Surg 2012;2:Doc06

DOI: 10.3205/gpras000008, URN: urn:nbn:de:0183-gpras0000087

Published: July 23, 2012

© 2012 Herold et al.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en). You are free: to Share – to copy, distribute and transmit the work, provided the original author and source are credited.


Abstract

Since Peer presented his “cell survival theory” it is assumed that the grafted tissue will be implemented in some way into the host tissue. This lead to a big effort to produce a fat graft that is as much viable as possible. Various studies have been performed to analyse viability of fat grafts prepared with different techniques or protocols. All viability tests in vitro permit a non invasive way to analyse human fat grafts. But the influence of processing due to the viability test protocol on viability of fat grafts cannot be totally excluded. Furthermore no normative values exist and we do not know if the test will really measure the real viability of the fat graft. Therefore an in vitro test can only be rated by the following characteristics: a) degree of tissue modification by the test itself, b) specificity on adipose tissue, c) ability to depict the relative rate of viable cells to the total cells of the graft. After all an ideal in vitro test still has to be invented, as all available tests do have their indications but also their disadvantages. Animal models offer in vivo analysis of fat survival, but results can not be transferred directly to humans. If autologous fat transplantation is performed for volume augmentation purposes the technique of lipofilling with the best volume survival rate and the least complications like oil cysts or calcifications would be superior.

Zusammenfassung

Viele Studien zur Bestimmung der Vitalität von Fetttransplantaten bei der autologen Fetttransplantation sind bereits unternommen worden. Seit der Beschreibung der „cell survival theory“ durch Peer geht man davon aus, dass das Fetttransplantat in gewisser Weise in den Empfängerorganismus integriert wird. Daher ist die Gewinnung von möglichst vitalem Fettgewebe von großer Bedeutung. Vitalitätstests ermöglichen eine nichtinvasive Evaluation von humanen Fettgewebstransplantaten, allerdings kann hierbei eine Beeinflussung der Vitalität durch den Test per se nicht ausgeschlossen werden. Normalwerte existieren nicht und die tatsächliche Vitalität kann nicht bestimmt werden. Daher können diese Tests nur anhand folgender Kriterien beurteilt werden: a) dem Grad der Gewebsveränderung durch den Test, b) der Spezifität für Fettgewebe, c) der Möglichkeit die relative Anzahl vitaler Zellen des Transplantates anzuzeigen. Ein idealer Vitalitätstest zur Fettgewebsdiagnostik muss noch entwickelt werden, alle bisherigen Tests haben entscheidende Vor-, und Nachteile. Tierversuche bieten die Möglichkeit der in vivo Diagnostik, aber die Ergebnisse können nicht direkt auf den Menschen übertragen werden. Falls eine autologe Fetttransplantation zur Gewebsaugmentation durchgeführt wird, kann diejenige Technik, welche die geringsten Komplikationsrate an Fettgewebsnekrosen oder Ölzysten bietet und den höchsten Volumenerhalt gewährleistet als am besten geeignet angesehen werden.


Introduction

Many studies have been performed on autologous fat transplantation. Common approaches to analyse viability of transplanted adipose tissue are animal studies or vitro settings in the laboratory with various viability tests. Since Peer presented his “cell survival theory” in 1950 [1] it is assumed that the grafted tissue will be implemented in some way into the host tissue. This lead to a big effort to produce a fat graft that is as much viable as possible. Although is has been shown that excised fat is more viable that fat grafts that have been gained by liposuction [2], today there is no question about the need for injection of small fat particles in multiple layers to minimize the risk of complications and to maximize the success of fat grafting in terms of graft survival [3], [4]. Therefore fat grafts obtained from liposuction are the standard procedure in autologous fat transplantation.

Various questions about possible factors that might affect viability of fat grafts have been addressed and their results have been published. The effect of the size of the suction cannula [5], [6], harvesting technique [7], [8], [9], [10], [11], [12], [13], [14], the applied suction vacuum [10], [15], harvest site [16], different local anaesthetics used in the tumescent solution [17], [18], [19], the effect of centrifugation [7], [9], [10], [16], [20], [21], [22], [23], [24], cryopreservation [6], [25], [26], [27] or special transfer media [28], [29] or even hyperbaric oxygenation [30] or the application of erythropoietin [31] have been analysed.


Animal models

Nude mice

Piasecki et al. described a model with genetically identical, age- and sex-matched mice to evaluate different harvest and preparatory techniques [12]. In 1998 Ullmann and colleagues injected fat into six weeks old. CD-1 male nude mice. The scalp was chosen as the recipient site for human fat, as no native subcutaneous fat is to be found in this area. This theoretically gives way for later dissection of plain human fat [29]. The weight and volume of the fat accumulation as well as histologic analyses by hematoxylin- and eosin staining can be performed. Based on this model, the effect of a transfer medium [29], hyperbaric oxygenation [30] and later erythropoietin [31] has been evaluated. Even if the nude mouse model presented by Ullmann [32] is promising because of the limited ability to reject xenogeny antigenic grafts and has been used in many other studies [19], [30], [33], [34], [35], [36] it is still an animal model and the results can not be completely transferred in humans. Furthermore weight and volume alone as well as hematoxylin-and eosin staining are no direct marker of viability. Eto successfully used a nude mouse model and contributed significant data in engraftment process analysis over the last years In nude mice the subcutaneous inguinal fat pad was elevated and small communicating vessels to the skin from the fat pad were dissected. This pad has a weight of 150 to 200 mg [33]. Ischemia was provocated by clamping the main vessels with a vessel microclip for 3 hours. It was then released to allow reperfusion. Adipose tissue was harvested at various time intervals [37]. In another mouse model setting, the groin fat pad was transplanted under the scalp, and the graft was stained at different timepoint up to 14 days. This in vivo study demonstrated that most adipocytes in the graft began to die on the first postoperative day, and only some of the adipocytes located within 300 µm of the tissue edge survived [33].

We think that the main disadvantage of a mouse model is the limited amount of adipose tissue so there might be a risk of miscalculation of survival rates. If it is used in a way like Eto and Suga did as described above, to analyse basic processes like engraftment process, it is a nice model.

Rats

Rats have an anabolic metabolism throughout their whole lifetime. That is why it is a common model [38]. In a study by Nishimura the fat was harvested from the inguinal region of the rat and 300 mg of fat was transplanted between the skin and the underlying muscle in the back [39]. In another rat animal model the epidimydal fat pad has been used, which is able to donate 2 g of fat. This fat was injected in small pearls in various regions of the same rat [40], [41]. Of those receptor regions the inguinal fat pad is most likely the region similar to the periglandular fat tissue, because it mainly consists of adipose tissue. Rieck [42] presented the use of PKH 26 (Zynaxis, Malvern, distributed by Sigma Immuno Chemicals, St. Louis, MO), an extremely lipophylic cell membrane marker that binds to the lipid layer of cell membranes for in vivo analyses of fat survival in rats [40], [41]. Is was invented by Horan and Slezak [43] and is an aliphatic molecule and can be coupled with various dyes, at best with rhodamine like dyes [40], [41]. The PHK 26 dye is said to have no influence on viability and mitosis rate of cells [43], however the necessity of digestion and sedimentation of fat tissue before staining might be harmful [40].

In 2006 Matsumoto et al. described the use of green fluorescent protein (GFP) rats [35]. The inguinal adipose tissue has been used in this study. The main advantage of using these rats as donor for fat transplantation is that the green fluorescent protein grafts will be easily discriminated from normal host tissue at time of harvesting and following immune histology. This model served very well to support the cell survival theory, which means that the transplanted fat graft is integrated in the host tissue. It further implies that the fat graft will survive in a certain amount and will be detectable in a long term follow up. It was presented by Peer [1].

Rabbits

Because it almost contains no adipose tissue, the ear of a rabbit seems to offer the chance to follow up the transplanted fat without interaction or mixing to local fat. Therefore this model has been advocated in earlier studies [15], [44], [45], [46] but also in actual studies [28]. On the other hand does the thin subcutanous plane above the cartilage like in the rabbit’s ear misrepresent the normal plane in autologous fat transplantation f.e. to the breast, for gluteal augmentation or for lip or face augmentation or hand rejuvenation [4] where either subcutaneous fat or muscles represent the receptor tissue [47].

All animal models (Table 1 [Tab. 1]) offer in vivo analysis of fat survival, but results can not be transferred directly to humans. Is has recently been shown in that adipose tissue is a superior receptor to fat transplants than f.e. muscle tissue in humans [48], where as it was shown the other way round in rats [49].

Furthermore the injected volume in all animal models reported has been very small. If the whole graft only contains a few grams or less of tissue, analysis of volume after resection of the graft after a while might be falsified, as measuring inaccuracy is hardly to avoid. Recipient sites differ from those of clinical fat transplantation as the animals used do not have breasts or comparable gluteal muscles.


In vitro viability testing

Morphological histological analysis

Analysing the plain morphology of fat grafts after fixation and hematoxilin-eosin and Mallory’s trichrome staining is a simple tool to evaluate the intactness of cells, the integrity and presence of mature nucleated adipocytes, cysts and vacuoles, fibrosis and inflammation [23], [24], [28] but does not give any information about the viability [26].

Histological analysis with trypan blue, sudan black or acridine orange staining

The trypan blue test is a membran integrity test [27]. After processing the adipose tissue specimen with collagenase at 37° staining with trypan blue vital stain is performed [5], [8], [9], [10], [12], [21], [50]. The digestion is necessary as the viability of clumps of adipose tissue is difficult to interpret [26]. The number of viable cells can be determined afterwards by microscopic examination. Besides the possible influence of collagenase digestion on cell viability this test relies on indirect viability measuring [16]. The wide range of results reported with this test may be conditional on the technical bias of analysis as well as the fact that dead cells might also be counted for viable cells, as they may appear normal under the microscope [27]. Furthermore lipid droplets cannot be distinguished easily from adipocytes because both are not stained very well [51]. Sudan black or acridine have been applied as well [52], [53], [54]. Although this method is well established, the are recommendations, not to use this staining in adipocytes [51].

Annexin V and/or propidium iodide

The Annexin V propidium iodide fluorescence stain can be used to distinguish between living and non living cells. It is a membran integrity test [27]. Propidium iodide is staining DNA and is being used as a marker for necrotic cells, as it is not able to stain viable or apoptotic cells due to the intact nucleus membrane. Annexin V is staining apoptotic cells as it binds to Phosphatidylserine which is not found on the cell membrane of intact cells. In apoptotic cells the cell membrane is everted and Phosphatidylserine is exposed. These stains are based on single cell suspensions. A fluorescence activated cell sorting (FACS) is necessary. This method uses a laser ray to count cells and various surface markers when they flow through a capillary [55]. Collagenase digestion is needed to produce the single cell solution, which as mentioned before, might interfere with cell viability. Applying the FACS analysis a broad variety of studies have been performed [17], [18], [56]. Alternatively cell count can be performed using a heamatocytometer under a microscope [27], [51]. Furthermore viability tests have been performed with propidium iodide fluorescence staining and ceiling culture [20], [26]. The combination of propidium iodide, and Hoechst 33342 to mark the nuclei, with AdipoRed staining allows differentiation between dead and viable adipocytes and non-adipocytes, like among the propidium iodide negative cells only viable adipocytes would be Hoechst 33342 positive and lipid containing (AdipoRed positive) [20], [51].

Colorimetric assays of cell viability

Colorimetric assays of cell viability are functional assays [27]. In mitochondria of vital cells a tetrazolium salt is being metabolized to a coloured water soluble formazin dye which is directly proportional to the living cells of the analysed tissue [16]. Analysis is performed with a microplate reader [27]. Where as the MTT and the XTT assays have been widely used for assessment of adipocite vability [7], [25], [27], the MTT assays has been shown not to produce reliable results as the formazan is insoluble and results may be poorly reproducible [51]. It has been shown in previous studies that the XTT assay is useful in quantification of adipocyte viability [7], [16], but as it is not specific for adipocytes [51]. The WST-8 test is a similar colorimetric assay. Alamar blue, based on resazurin, has also been used to analyse adipose cell viability and is reduced in metabolically active cells to red fluorescent dye, which fluorescence again is proportional to the number of living cells [13]. The main advantage of colorimetric assays of cell viability is that a collagenase ingestion is not needed to produce the single cell soulution and that the protocols do not contain centrifugation steps, which might be especially important, when analysing the influence of centrifugation during clinical fat transplantation. Not producing single cell solutions and staining intact tissue pieces on the other hand givens way to the possibility to just stain superficial parts of the tissue. Diffusion to central parts of the tissue is a limiting factor for functional assessment of 3 D united cell structures. Further more the total cell number cannot be analysed and viability can only be defined in relation to another base line tissue.

GPDH assay

The glycerol-3-phosphatase dehydrogenase (GPDH) assay is a membrane integrity test and detects escaped intracellular enzyme passed through the damaged cell membrane [27]. GPDH is a cytosolic enzyme found specifically in adipocytes and can be found in the extracellular medium if the cell membrane of adipocytes is mechanically damaged [13], [27]. The accuracy of this viability test is affected by the fact that not only the extracellular GPDH of damaged cells, but also the intracellular GPDH of viable cells is measured [51]. Only the extracellular GPDH should be considered proportional to cell destruction and cell counting of adipocytes and it would be necessary to correlate the GPDH values and the vital adipocytes [51]. Some authors describe the protocol of this test to be quite complicated [23], where as other authors accentuate that it is very simple [10], [25]. Another advantage of the GPDH test is that the production of a single cell solution is not necessary, which eliminates one possible step to falsify the results [25].

Glucose transport test

Based on the finding that viable adipocytes transport glucose into the cell to maintain metobolism, quantification of glucose within the adipocyte can be used to identify viable adipocytes [23]. Xie et al. describe that this test analyses the viability of the adipose tissue graft in total and not only of single adipocytes and therefore thinks it is useful to evaluate the viability of the fat graft. Until now is not clear if elevated fat graft glucose does really represent elevated fat graft viability.

DNA assays

PicoGreen DNA assay is a sensitive fluorescent nucleic acid stain for quantitating double-stranded DNA in solution and serves as a proliferation assay and offers the possibility to test whether the cellular proliferation is changed in fat grafts. It is based on counting the total DNA content of a probe. It is not specific for adipose cells but an advantage is that diffusion to central parts of the tissue is not a problem like it may take place in metabolic assays. PicoGreen DNA assay was used to find out that liposuction causes more damage to fat tissue than scraping, but it offers the chance for better long-term viability based on increased proliferation [23].

Brilliant cresyl blue zinc chloride double salt staining works in a similar way by staining the desoxyribonucleotic acid of viable cells [6]. Again the tissue has to be digested by a collagenase before staining.

In situ labelling of apoptotic cells using the TUNEL test

Using a commercially available in situ detection kit apoptosis can be detected by labelling the 3'OH ends of the DNA by digoxogenin incorporation applying the terminal deoxynucleotidyl teranferase (TdT)-mediated desoxy-uridine-triphosphate (dUPT) – biotin nick end labelling method [39]. Nishimura used the Apop Tag kit [39]. The TUNEL test is not specific for adipose tissue but in can detect apoptosis of the fat graft, consisting of various cell types [41], in general.

Perilipin staining

Perilipin marks lipid droplets only in viable adipocytes, dead adipocytes can not be stained with perilipin [57]. Eto et al. used this method as it is specific for viable adipocytes, which appear as round-shaped cells that are strongly positive for perilipin [33]. Using this technique the authors have been able to demonstrate that almost all adipocytes located deeper than 300 mm away from the superficial surface of the fat graft died within a few days after grafting. Adipocytes were the first cells of fat graft recognized to die on the first day after ischemia. They were followed by endothelial cells and finally adipose-derived stromal cells died on day three [33]. It is an important study, because it may lead to a paradigm shift, giving up the above mentioned „cell survival theory“.


Conclusions

All viability tests in vitro (Table 2 [Tab. 2]) offer a non invasive way to analyse human fat grafts. This is in particular interesting, as fat grafts harvested by liposuction have been waste for many years, and still are if not an autologous fat transplantation is performed. Availability of tissue to perform these tests is very high although liposuction itself is an invasive procedere. There is no inter-species bias. But with all staining methods, the influence of processing on viability cannot be totally excluded. As no normative values exist and we do not know if the test will actually measure the real viability of the fat graft or only something the test is able to measure. A suitable in vitro test should cause a minimal degree of tissue modification, it should be at best be specific on adipose tissue and it should offer the possibility to display the relative rate of viable cells to the total cells of the graft. Most of the available tests are based on protocols that include centrifugation steps like the Annexin V propidium iodide [18] or the GPDH [51] test. Especially in studies analysing the effect of centrifugation forces on fat viability colorimetric tests like the XTT or WST-8 test seem to be useful as they do not include centrifugation steps. Furthermore digestion of the fat graft to produce a single cell suspension before viability testing in trypan blue [26] or Annexin V propidium iodide [18] might also interfere with adipose tissue viability. The superior specificity of the GPDH test for adipose tissue would make this test eligible to be the standard test for adipose tissue analyses, if there was not the problem the intracellular GPDH in viable cells that will be measured as well. Annexin V propidium iodide tests are conveniently incorporated in surface marker protocols in FACS analysis that also analyse adipose derived stem cell (ASCs) content and is furthermore not examinator dependent like trypan stained cell counting as is can be performed automatically by cell coulters. The possibility of relative cell viability in comparison to the whole graft is at best given in FACS based propidium iodide staining or trypan blue staining, as the number of viable cells can be easily compared to the number of non viable cells. In GPDH or colorimetric tests a reference tissue has to be analysed as well (for example plain excised fat).

After all the ideal in vitro test still has to be invented, as all available test do have their indications but also their disadvantages.

Animal models offer the chance of in vivo analyses without harming humans. Nevertheless results can not be transferred directly to the clinical situation of autologous fat transplantation in humans as initial situation differs too much.


Notes

Meeting presentation

Parts of this review have been presented by the first author at 128. Kongress der Deutschen Gesellschaft für Chirurgie, 03.05.–06.05.2011, Munich, Germany.

Competing interests

The authors declare that they have no competing interests.


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