- Email: [email protected]
stromal cells
Journal of Plastic, Reconstructive & Aesthetic Surgery (2013) 66, 1271e1278
Conventional vs. micro-fat harvesting: How fat harvesting technique affects tissue-engineering approaches using adipose tissue-derived stem/stromal cells ¨nder a,d, Sultan Almakadi a, Ziyad Alharbi a,b,*,d, Christian Opla Andrea Fritz a, Michael Vogt c, Norbert Pallua a Department of Plastic, Reconstructive and Hand Surgery e Burn Center, Medical Faculty, RWTH Aachen University, Pauwelsstr. 30, D-52074 Aachen, Germany b Division of Plastic and Reconstructive Surgery, Department of Surgery, King Abdullah Medical City, Kingdom of Saudi Arabia c Two-Photon Imaging Core Facility, Interdisciplinary Center for Clinical Research (IZKF), Medical Faculty, RWTH Aachen University, Pauwelsstr. 30, D-52074 Aachen, Germany a
Received 11 November 2012; accepted 10 April 2013
KEYWORDS Adipose tissuederived stem/stromal cells; Stromal vascular fraction; Liposuction; Lipofilling; Fat grafting; Biomaterials; Collagen-based scaffolds
Summary Background: Biocompatible scaffolds as dermal substitutes are used commonly in soft tissue reconstruction and tissue-engineering approaches. The combination of these scaffolds with mesenchymal stem and stromal cells would have additional benefits in multilayer soft tissue reconstruction. In addition, the use of lipoaspirate may be beneficial for this purpose containing high levels of regenerative cells and relevant growth factors. However there are many factors, which may impact the lipoaspirate content of isolated cells, cell behaviour and growth factors. There is a lack of data as to whether fat-harvesting procedures using different cannulas of small diameter will impact these parameters, which are relevant not only for tissue engineering but also for clinical outcome. Methods: Abdominal liposuctions were performed on 10 patients using the conventional fat harvesting by the Coleman cannula (3 mm, one-hole blunt tip) and the micro-fat-harvesting technique by the st’RIM cannula (2 mm, multi-perforated hole blunt tip) on contralateral area. Lipoaspirate contents of insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) were measured by enzyme-linked immunosorbent assay (ELISA). The in vitro viability of
* Corresponding author. Department of Plastic, Reconstructive and Hand Surgery e Burns Unit University Hospital of the RWTH Aachen University, Pauwelsstr. 30, 52057 Aachen, Germany. Tel.: þ49 241 80 0; fax: þ49 241 80 82634. E-mail address: [email protected] (Z. Alharbi). d Both authors contributed equally to this work. 1748-6815/$ - see front matter ª 2013 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bjps.2013.04.015
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Z. Alharbi et al. lipoaspirates was tested by the alamarBlue assay. Adipose-derived stem/stromal cells (ASCs) were isolated and the yields determined. Furthermore, ACSs were seeded on collagen elastin matrices (Matriderm) and cell migration/adhesion rate was examined by the alamarBlue assay and visualised by two-photon microscopy. Results: Conventionally obtained lipoaspirates were found to contain significantly higher concentrations of IGF and VEGF, but not PDGF or bFGF. No significant effects on the yields of ASCs or the in vitro viability of lipoaspirates obtained from different cannula sizes were observable. However, the viability and migration of isolated ASCs obtained from micro-harvested lipoaspirates were significantly higher. Moreover, a significant high adherence rate of isolated ASCs from the micro-fat-harvesting technique onto matrices was observed. Conclusion: The different sizes and surface/volume ratios of pieces of fatty tissue obtained by using different cannula sizes may be responsible for the observed differences and effects. Thus, micro-fat harvesting may be more suitable for tissue-engineering and -regenerative approaches using ASCs and collagen elastin matrices. ª 2013 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved.
Soft tissue augmentation by lipofilling with adipose tissue derived from liposuction procedures has shown promising results with less invasive techniques, but variation in longterm outcome in terms of loss of transplanted volume has been reported.1e3 One reason for a decreasing volume is insufficient blood supply after transplantation. Therefore, angiogenesis process is crucial for the survival of adipocytes, which are subject to apoptosis and necrosis if not supported by a network of capillary vessels by the fourth day after implantation.4 Studies have shown that the plasmatic nutritional supply of adipocytes can only go as far as 1.5 mm into tissues5 and that only 40% of the fat at a distance of 1.5 mm from the boundary of the tissue block survives.6 Growth factors, in particular, vascular endothelial growth factor (VEGF), are essential for angiogenesis, and after a short latency period the sprouting vessels organise into a capillary blood supply network, enabling adipocytes to survive.7 Lipoaspirates, obtained from liposuction, contain VEGF and many other growth factors, including basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF) and platelet-derived growth factor (PDGF).8 In animal studies, these growth factors have been shown to improve transplantation results.9e12 In the adipocyte-free fraction of lipoaspirate, the stromal vascular fraction (SVF) contains a variety of cells such as pre-adipocytes, endothelial cells, smooth muscle cells, pericytes, fibroblasts, macrophages and adult stem cells.13 Under certain conditions, these cells, called adipose-derived stem/stromal cells (ASCs), can be induced to differentiate into not only adipocytes but also bone, neuronal or endothelial cells. Therefore, ASCs, and especially the fraction of stem and progenitor cells, present an innovative option for regenerative cell therapy.14,15 For example, ASCs have been shown to play a major role in promoting wound healing16,17 and secrete almost all growth factors involved in normal wound healing.18 Furthermore, it has been reported that ASCs promote vessel density, the granulation process and collagen thickness16 and thus may improve the cosmetic aspect of the resultant scar.18 Since fatty tissue is a major source of stem cells19 and growth factors, the use of fatty tissue may provide more clinical benefits than augmentation alone.
However, studies have shown that apart from sex and depot-dependent differences,20e22 the yield of stem cells isolated from lipoaspirates can be affected by the fatharvesting procedure and/or lipofilling techniques.23 Furthermore, the combination of isolated adipose tissue derived cells with collagen-based scaffolds would provide another dimension in soft tissue reconstruction through tissue-engineering approaches. Clinically, collagen elastin matrices (Matriderm) have been used in multistage operations or even in one-stage operations as they have been cooperated with autologous split thickness skin transfer for soft tissue reconstruction. Variables such as the ideal cannula and technique for harvesting, the best way of processing fat to ensure maximum uptake and viability of the graft and ACS on collagen elastin matrices, are all factors which require clarification.2 For using fresh-isolated ACSs for tissueengineering or -regenerative approaches the harvesting technique may be crucial, because cells have to adhere fast onto scaffolds to avoid ‘cell slipping’ away from scaffold during transplantation or from the original site of recipient area after transplantation. Also factors, such as cell yield, viability and growth factors, influenced by the harvesting technique may affect the performance of ACS-based tissue engineering for regenerative medicine purposes. Therefore, the aim of this study is to compare the influence of the fat-harvesting procedures (conventional vs. micro-fatharvesting technique) on the viability and content of growth factors of the grafts obtained. Furthermore, we have investigated the effects of both methods on the yield, migration and adhesion rate of the ASCs which have been isolated directly from the corresponding lipoaspirates.
Methods and materials Patients With the patients’ consents, lipoaspirates were obtained from 10 healthy patients (five males and five females; age range 27e59) having undergone an elective liposuction procedure
Conventional vs. micro-fat harvesting from a single anatomical site (abdominal subcutaneous fat tissue) at the Department of Plastic and Hand Surgery e Burns Centre, RWTH Aachen University Hospital. Fat tissues have been obtained from the deep layer by the same surgeon (NP). Patients with body mass index (BMI) >30, diabetes, hypertension and nicotine or alcoholic abuse have been excluded. The protocol and use of lipoaspirates were approved by the regional ethics committee (EK163/07) and conducted in compliance with the Declaration of Helsinki Principles.
Liposuction procedures Conventional fat-harvesting technique This established method was performed as described by SR Coleman.24 A mixed solution (1 liter of sodium chloride 0.9%, and 12.5 ml of prilocaine 1%, and 1 ml of epinephrine 1:200,000) was infiltrated into the donor site. The solution was infiltrated at a ratio of 1 ml of solution per cubic centimetre. The fat grafts were harvested through the same incisions. The harvesting cannula was 3 mm in diameter with a single-hole blunt tip (Byron Medical Inc., Tucson, AZ, USA) connected to a 10-ml syringe (see Figure 1). After filling the syringe with harvested tissue (20 cc), the cannula was removed from the syringe. Micro-fat-harvesting technique The procedure as outlined above was repeated in the contralateral region to obtain another 20 cc of harvested fat. However, in contrast to the Coleman cannula, the used harvesting cannula was an st’RIM cannula (Thiebaud Biomedical Devices, Margencel, France) developed by Guy Magalon for micro-lipografting. This cannula was 2 mm in diameter with a blunt tip and four 600-mm-gauged orifices (see Figure 1) (for details, see http://www.mystrim.com/surgeon/).
Washing and centrifugation of the obtained lipoaspirate With each patient, the two different liposuction techniques were performed. Immediately after the liposuction procedure, the body of the filled syringe was placed into a centrifuge (Sigma 2e16 K, Osterode am Harz, Germany) and centrifuged (3 min, 300 rpm). After centrifugation, the oil layer (upper level) was decanted and the aqueous layer (lower level) was drained from the syringe. The middle layer, composed of predominantly fat grafts, represents the purified lipoaspirate, which was used immediately for experiments and for cell isolations.
Isolation of SVF cells from lipoaspirate From each patient with special consideration to the selected fat-harvesting procedure, a volume of 20 ml of purified lipoaspirate, from each method, was transferred into a sterile
Figure 1 Shows the 2 mm st’Rim harvesting cannula (above) and the 3 mm Coleman harvesting cannula blunt type (below).
1273 tube and 30 ml of normal saline was added. Then, a second centrifugation process was completed for 10 min at 300 rpm. After centrifugation, adipose tissue was transferred into a sterile tube for the isolation process. Collagenase solution was freshly prepared (1% bovine serum albumin (PAA, Linz, Austria) and 2% collagenase type 1 (Biochrom, Berlin, Germany)) in a buffer solution (100 mM 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES), 120 mM NaCl, 50 mM KCl, 1 mM CaCl2, 5 mM glucose, pH 7.4) and then was added to the centrifuged adipose tissue through a 0.2-mm filter under sterile conditions (enzyme/tissue ratio is 1:1). After incubation (45 min, 37 C) and under constant shaking, the digested tissue solution was filtered using a 250-mm filter (Neolab, Heidelberg, Germany) and centrifuged for 10 min at 300 g. Yellow rigid fatty tissue and supernatant were discarded and the pellet was resuspended with 30 ml NaCl solution and then was re-centrifuged at 300 g for another 10 min. The pellet was resuspended in DMEM/F12 cell culture medium (Invitrogen, Darmstadt, Germany) supplemented with 1% penicillin and 1% streptomycin (PAA, Linz, Austria) without further proliferation factors.
Determination of the content of isolated ASCs Isolated cells were counted manually under a light microscope using a Neubauer chamber (VWR, Darmstadt, Germany). A trypan blue assay made it possible to exclude dead cells. Then 10 mm trypan blue (PAA, Linz, Austria) was added to 10 ml of cell suspension before counting. Experiments using ASCs started directly after isolation.
Testing fat tissue viability and survival rate in vitro In parallel, purified lipoaspirates obtained from conventional fat-harvesting or micro-fat-harvesting procedures were transferred into a six-cell culture plate (1 g for each well) and 2 ml of DMEM/F12 cell culture medium supplemented with 1% penicillin/1% streptomycin/10% foetal calf serum added to each well. For testing the viability of lipoaspirates within the first 24 h after liposuction, 300 ml of alamarBlue reagent (AbD Serotec, Oxford, UK) was added into some wells and after 24 h incubation time (5% CO2, 37 C). The 2 100 ml Medium/alamarBlue mix was carefully removed from the well and immediately measured at room temperature using a fluorescence spectrometer (Fluostar Optima; BMG Labtech, Offenburg, Germany; excitation wavelength 540 nm, emission wavelength 590 nm).25 For testing the viability of lipoaspirates at 24 and 48 h after liposuction, 300 ml alamarBlue reagent was now added to the other wells and incubated for further 24 h (5% CO2, 37 C). After incubation, 2 100 ml Medium/alamarBlue mix was carefully removed from the well for fluorescence measurement.
Measurement of growth factors and cytokines in lipoaspirate fat tissue Purified lipoaspirate (1 g) was frozen immediately at 80 C. To evaluate the content of growth factors by enzyme-linked immunosorbent assay (ELISA), the samples were thawed on ice at 4 C, and 3 ml of homogenisation buffer (pH 7.5) was added to each sample. An Ultra-Turrax
1274 homogeniser (IKA Works, Inc., Wilmington, NC, USA) was applied twice for 30 s and the homogenised suspension was centrifuged (100 g, 10 min, 4 C). The supernatant was again centrifuged (20,000 g, 40 min, 4 C) and the oily phase and cell pellet were discarded. The supernatant obtained was divided into aliquots, shock-frozen in liquid nitrogen and stored at 80 C. To measure the content of bFGF, IGF-1, VEGF and PDGF-BB in the centrifuged lipoaspirate obtained from the conventional fat-harvesting technique and micro-fat-harvesting procedures, sample aliquots were analysed using appropriate ELISA duo sets (ELISA, R&D Systems, Minneapolis, MN, USA). Extinction was measured as recommended by the manufacturer using a FLUOstar OPTIMA microplate reader at 450 nm, and by using a benchmark value of 540 nm.
Incubation of Matriderm with ASCs or purified lipoaspirate to determine cell migration and adherence rate Matriderm sheets (1 mm thick) consisting of non-crosslinked native bovine collagen matrix (type I, III and V collagen derived from bovine skin) were used in this study (supplied by Dr. Otto Suwelack Skin and Health Care GmbH, Billerbeck, Germany). Circular pieces of Matriderm were obtained using a circular punch biopsy device measuring 0.8 cm, and then placed on 46-well culture plates. Custommade sterile tubes (diameter 0.8 cm) were used to fix the sheets and to avoid direct contact between cells and the bottom of the cell culture wells. Fat graft (1 g) was directly transferred into 46-well cell culture plates lined with Matriderm and incubated at 37 C and 5% CO2 for 24 h. Alternatively ASCs were directly added at a density of 5 104 (270 ml DMEM/F12) into 48-well culture plates lined with Matriderm and incubated at 37 C and 5% CO2 for 24 h in DMEM/F12 cell culture medium supplemented with 1% penicillin and 1% streptomycin with no proliferation factors.
Z. Alharbi et al. Japan, attached to a pulsed Ti-Sapphire laser from MaiTai DeepSee, SpectraPhysics). The collagen elastin matrix was visualized by the non-linear optical effect of second harmonic generation (SHG). For excitation of FDA and SHG the laser was tuned to the wavelength of 850 nm. The emission of FDA and SHG was collected at 495-540 nm and 418-468 nm respectively. Consecutively Hoechst was excited at 730 nm and detected at 418-468 nm. Series of subsequent xy-frames with 1024 1024 pixels in 1 mm z-steps were obtained for structural 3D reconstruction conducted with Imaris Software (Bitplane, Zurich, Switzerland).
Results Comparison of cell yields successfully isolated from conventional and micro-fat-harvesting techniques From lipoaspirates gained from either conventional or micro-fat-harvesting techniques, ASCs were isolated and
Determination of the number of isolated cells adhered at the matrix The number of viable ASCs was tested by using the alamarBlue assay. The Matriderm sheet, incubated with purified lipoaspirate or isolated ASCs, was separated after the 24-h incubation period. The sheet was washed carefully with normal saline (0.9% NaCl) and transferred into a clean culture plate. After suspension using 270 ml of pre-adipocyte medium (DMEM/F12 supplemented with 100 U ml1 penicillin and 100 mg ml1 streptomycin) with no proliferation factors, 30 ml of alamarBlue reagent was added to each well. After incubation for 2 h (37 C, 5% CO2), samples were measured directly at room temperature by a fluorescence spectrometer (excitation wavelength 540 nm, emission wavelength 590 nm).
Two-photon microscopy For vital staining of the isolated cells and visualisation of the adherent cells on the Matriderm, fluorescein diacetate (FDA) and Hoechst 33342 were added. The evaluation was carried out in just 24 h after seeding of the ASCs using a twophoton microscope (FV1000MPE, Olympus Corp., Tokyo,
Figure 2 A shows the quantification of the numbers of cells (ASCs) which could be isolated from lipoaspirates obtained by conventional and by micro-fat harvesting technique (20 g) under standard isolation procedure. B shows the concentration of Basic Fibroblast Growth Factor (bFGF), Insulin-like Growth Factor 1 (IGF-1), Vascular Endothelial Growth Factor (VEGF) and Platelet-derived Growth Factor (PDGF) found in homogenates of purified lipoaspirates obtained by by conventional and by microfat harvesting determined by ELISA; n Z 10; *p < 0.05.
Conventional vs. micro-fat harvesting
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yields were determined. Mean values are shown in Figure 2A. There were no significant differences in cell yields between the two techniques and standard deviations were in the same range, indicating strong intra-individual variations. However, the mean value for the micro-fatharvesting technique (3.60 107 cells g1 lipoaspirate) appears somewhat greater than the value from the conventional technique (2.94 107 cells g1 lipoaspirate).
Determination of growth factors of purified lipoaspirates Purified lipoaspirates from either the conventional or micro-fat-harvesting technique were homogenised and the concentration of growth factors relevant for cell growth and angiogenesis, such as bFGF, IGF-1, VEGF and PDGF, was determined by ELISA. As shown in Figure 2B, the concentrations of IGF-1 and VEGF found in both types of lipoaspirates were significantly different. The IGF-1 concentration of conventional lipoaspirates was about twice as great as the concentration of lipoaspirates obtained from the micro-fat-harvesting technique (192 128 vs. 77 83 pg mg1). Furthermore, significant differences were found in the VEGF concentrations, where conventional lipoaspirates were found to contain 52 29 pg mg1 against a figure of only 35 18 pg mg1 for the lipoaspirates obtained from the micro-fat-harvesting procedure. No significant differences were found in relation to bFGF and PDGF-BB concentrations.
Comparison of in vitro vitality of conventional and micro-fat grafts By using the alamarBlue assay, it was possible to carry out an estimation of living cells and fat graft vitality in vitro. After 24 h, the alamarBlue fluorescence values indicate the presence of living cells in the fat grafts. In our investigation, the mean signal relating to fat grafts obtained from the conventional technique was slightly, but not significantly, higher than the grafts obtained from the micro-fat-harvesting technique (Figure 3A). After 48 h, a significant decrease in fluorescence values was observed for both techniques, indicating a loss of cell vitality (38% conventional vs. 26% mico-lipografting) in the fat grafts.
Determination of lipoaspirate cell/ASC migration and adherence onto collagen matrices To determine the cell migration from lipoaspirates onto a collagen matrix, Matriderm sheets were incubated with lipoaspirates obtained from conventional and micro-fatharvesting techniques respectively, and the relative cell number of adherent cells was measured by the alamarBlue assay after 24 h. As shown in Figure 3B, significant, twice as high alamarBlue fluorescence signals were observed on Matriderm sheets incubated with lipoaspirates obtained by the micro-fat-harvesting technique (4311 1553 AU) than those obtained by the conventional technique (2071 747 AU). As shown in Figure 3C, with fluorescence values normalised to the cell number determined in the lipoaspirate in each instance and the
Figure 3 A shows the in vitro vitality of purified lipoaspirates obtained by conventional and by micro-fat harvesting in 24 h and 48 h after liposuction, B shows the number of cells adhering to the Matriderm collagen sheets after 24 h incubation with purified lipoaspirates. C shows values normalised to the cell number determined in the used lipoaspirate in particular. Mean and standard deviation from 6 to 10 independent experiments are provided; *p < 0.05.
difference between the numbers of translocated cells remains significant (micro-lipografting 11.95 6.69 AU vs. conventional lipografting 7.13 2.87 AU normalised to 50,000 cells), indicating that the greater cell number found in micro-fat-harvesting procedure is not the only reason for the higher migration and adherence rate of cells.
1276 Furthermore, there is also a significant difference in the adhesion rate between freshly isolated ASCs from lipoaspirates obtained by the two different techniques. Here, as shown in Figure 4A, Matriderm sheets incubated for 24 h with a defined number of cells isolated from the micro-fatharvesting technique showed significantly higher fluorescence values than those obtained by the conventional technique (6537 3667 AU vs. 2892 1547 AU). Moreover, the exemplary visualisation of the Matriderm sheets using two-photon microscopy shows that the seeded isolated adipose-derived cells could be found within the Matriderm collagen matrix after a 24-h incubation time (Figure 4B).
Discussion Today, autologous fat grafting is a beneficial procedure for patients with volume loss in reconstructive and cosmetic
Figure 4 A shows the alamarBlue fluorescence values, which are proportional to number of viable cells on Matriderm sheets incubated for 24 h with isolated ASCs obtained by conventional and micro-fat harvesting technique. Mean and standard deviation of 6 independent experiments are provided; *p < 0.05. B shows exemplary 3-D visualisation of ASCs which were adherent after 24 h incubation of the Matriderm sheet with isolated ASCs.
Z. Alharbi et al. surgery. However, the resorption rate is seen to be a variable and the loss of volume may require further grafting procedures.26 One point for consideration is the method of ¨ zsoy et al. stated that the use of widerliposuction. O diameter cannulas can potentially improve fat graft survival and reduce fat graft resorption.27 One important characteristic of adipose tissue and adipose-derived stem/ stromal cells is the secretion of a variety of growth factors and cytokines,8 which can be beneficial for patients. For example, using nude mice as a model, Mojallal et al. found a local increase of type I collagen fibres of murine origin after injection of human fatty tissue resulting from murine fibroblast stimulation by the human fatty tissue. Additionally, dermal thickness significantly increased around the injection area.28 In this study, we used the conventional Coleman technique (3-mm-diameter blunt-type cannula) and the micro-fat-harvesting technique (2-mm-diameter blunt-type cannula with multiple perforated orifices of 600 mmgauged) on the same patients in the abdominal region only with the same centrifuge power and time, thus allowing us to focus on the impact of cannula size on graft viability, ACS yield, growth factor concentrations and cell behaviour. We observed slight, but not significant, differences in the yields of ASCs (Figure 2). Under application of the same isolation procedure, lipoaspirates obtained by the micro-fatharvesting technique yielded cell numbers 22.4% higher than obtained with the conventional technique. However, differences in cell numbers between patients were high, resulting in high standard deviations. Significantly, higher VEGF and IGF-1 concentrations (but not bFGF and PDGF-BB) were found in conventional obtained lipoaspirates. By using a smaller cannula size (st’RIM) with small 600-mm-gauged orifices, lipoaspirates with smaller tissue pieces are obtained, where the surface/volume ratio is higher compared with larger tissue pieces obtained by a larger cannula and size (Coleman technique), leading to an increased loss of secreted soluble of VEGF and IGF-1 during the washing steps of lipoaspirate purification. Since the development of bFGF and PDGF-BB is more intracellular, their total concentrations would be impacted by the cell numbers in the pieces of tissue rather than by the surface/volume ratio and washing steps, which in our case are not significantly different. A significant loss of vitality after 48 h was identified in conventional lipoaspirates. However there was also a loss of vitality in the case of the micro-fat-harvesting lipoaspirates, indicating that it was not significant. A possible reason for this difference may be a reduced oxygen supply via diffusion for the larger pieces of tissue of the grafts obtained by conventional liposuction. However, a recent study from Nguyen et al. in 2012 showed that following fat transplantation into mice, the viability and volume of fat grafts obtained using a classical 3-mm Coleman harvesting cannula were not significantly different in comparison to fat grafts obtained by multiperforated cannula with holes of 600 mm-gauged in diameter.29 Furthermore, both injected fat grafts maintained a normal histological structure. Apart from clinical aspects, for tissue-engineering and -regenerative approaches the yields, viability and cell behaviour of ASCs freshly isolated from lipoaspirates can be impacted by many parameters, such as harvesting procedure.23 In such cases, for optimal results, the ideal cannula
Conventional vs. micro-fat harvesting and technique for harvesting, and also the best way and time of processing the fat graft for cell isolation have yet to be identified.2 In our study we could observe that the harvesting technique influences the ASC migration significantly. Here, ASCs migrate more likely form micro-fat grafts to a collageneelastin matrix (Matriderm) than conventional grafts. This may be important for enriching scaffolds with ASCs for tissue engineering without further processing steps for cell isolation and culturing. The higher surface/volume ratio of fatty pieces of tissue in lipoaspirates obtained from the micro-fat-harvesting technique could explain the results observed. In an in vivo situation, a higher migration rate from cells of the graft to the existing intracellular matrix in the recipient area may provide beneficial effects for the patients. In addition, the finer lipoaspirates obtained by micro-fat harvesting allow an application onto the superficial layer of the skin using smaller cannulas, which would be clogged by conventional lipoaspirates. Interestingly, after cell isolation, the adhesion rate of ASCs from micro-fat harvesting to the collagen matrix is twice as high as that of ASCs obtained from the conventional liposuction technique. This may be explained by differences in cell populations obtained after isolation. Moreover, the observed differences in the concentration of growth factors in lipoaspirates may impact the adhesion response of ASCs. To recapitulate, the study has shown that the technique and the cannula used for liposuction can impact the number of cells and the viability of the lipoaspirate tissue. Furthermore, the liposuction technique used has a significant impact on growth factor concentrations of purified lipoaspirates, cell migration from pieces of fatty tissue and also the adherence rate of ASCs. The probability is that these observed effects are due to the different sizes and surface/volume ratios of pieces of fatty tissue obtained using different cannula sizes. Such results are important not merely for a scientist in the bench side but also for clinicians who want to drive more viable isolated cells into a clinical tissue-engineering approach for multilayer soft tissue reconstruction in an autologous approach. We advance the hypothesis that a minimal fat lobule size not only is required for graft survival after lipofilling but also may represent an important factor for adipose-derived stem cell-based approaches. Nevertheless, further experimental studies and clinical trials have to be conducted to support such an assumption.
Conclusion The different sizes and surface/volume ratios of pieces of fatty tissue obtained by using different cannula sizes may be responsible for the observed differences and effects. Thus, micro-fat harvesting may be more suitable for tissueengineering and -regenerative approaches using ASCs and collageneelastin matrices.
Declaration The use of human material was approved by the local ethics committee of the Medical Faculty of the RWTH University
1277 Aachen (Number: EK163/07) and conducted in compliance with the Declaration of Helsinki Principles.
Disclosures Authors state no disclosures.
Conflict of interest The authors state no conflict of interest.
Acknowledgements We thank Dr. Otto Suwelack Skin and Health Care GmbH for supplying the Matriderm sheets. This work was partially supported by the Two-Photon Imaging Core Facility for imaging process, a core facility of the Interdisciplinary Center for Clinical Research (IZKF) Aachen within the Faculty of Medicine at RWTH Aachen University.
References 1. Boschert MT, Beckert BW, Puckett CL, Concannon MJ. Analysis of lipocyte viability after liposuction. Plast Reconstr Surg 2002; 109:761e5 [discussion 66e7]. 2. Bucky LP, Percec I. The science of autologous fat grafting: views on current and future approaches to neoadipogenesis. Aesthet Surg J 2008;28:313e21 [quiz 22e4]. 3. Sommer B, Sattler G. Current concepts of fat graft survival: histology of aspirated adipose tissue and review of the literature. Dermatol Surg 2000;26:1159e66. 4. Crandall DL, Hausman GJ, Kral JG. A review of the microcirculation of adipose tissue: anatomic, metabolic, and angiogenic perspectives. Microcirculation 1997;4:211e32. 5. Cortese A, Savastano G, Felicetta L. Free fat transplantation for facial tissue augmentation. J Oral Maxillofac Surg 2000;58: 164e9 [discussion 69e70]. 6. Carpaneda CA, Ribeiro MT. Percentage of graft viability versus injected volume in adipose autotransplants. Aesthetic Plast Surg 1994;18:17e9. 7. Yamaguchi M, Matsumoto F, Bujo H, et al. Revascularization determines volume retention and gene expression by fat grafts in mice. Exp Biol Med (Maywood) 2005;230:742e8. 8. Pallua N, Pulsfort AK, Suschek C, Wolter TP. Content of the growth factors bFGF, IGF-1, VEGF, and PDGF-BB in freshly harvested lipoaspirate after centrifugation and incubation. Plast Reconstr Surg 2009;123:826e33. 9. Schweigerer L. Fibroblast growth factor and angiogenesis. Z Kardiol 1989;78(Suppl. 6):12e5. 10. Eppley BL, Sadove AM. A physicochemical approach to improving free fat graft survival: preliminary observations. Aesthetic Plast Surg 1991;15:215e8. 11. Bluher S, Kratzsch J, Kiess W. Insulin-like growth factor I, growth hormone and insulin in white adipose tissue. Best Pract Res Clin Endocrinol Metab 2005;19:577e87. 12. Hausman GJ, Richardson RL. Adipose tissue angiogenesis. J Anim Sci 2004;82:925e34. 13. Witkowska-Zimny M, Walenko K. Stem cells from adipose tissue. Cell Mol Biol Lett 2011;16:236e57. 14. Mizuno H, Tobita M, Uysal AC. Concise review: adipose-derived stem cells as a novel tool for future regenerative medicine. Stem Cells;30:804e10.
1278 15. Gimble JM, Bunnell BA, Chiu ES, Guilak F. Concise review: adipose-derived stromal vascular fraction cells and stem cells: let’s not get lost in translation. Stem Cells;29: 749e54. 16. Ebrahimian TG, Pouzoulet F, Squiban C, et al. Cell therapy based on adipose tissue-derived stromal cells promotes physiological and pathological wound healing. Arterioscler Thromb Vasc Biol 2009;29:503e10. 17. Tobita M, Mizuno H. Adipose-derived stem cells for periodontal tissue regeneration. Methods Mol Biol;702:461e70. 18. Blanton MW, Hadad I, Johnstone BH, et al. Adipose stromal cells and platelet-rich plasma therapies synergistically increase revascularization during wound healing. Plast Reconstr Surg 2009;123:56Se64S. 19. Zuk PA, Zhu M, Ashjian P, et al. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell 2002;13: 4279e95. 20. Padoin AV, Braga-Silva J, Martins P, et al. Sources of processed lipoaspirate cells: influence of donor site on cell concentration. Plast Reconstr Surg 2008;122:614e8. 21. Fraser JK, Wulur I, Alfonso Z, Zhu M, Wheeler ES. Differences in stem and progenitor cell yield in different subcutaneous adipose tissue depots. Cytotherapy 2007;9:459e67. 22. Tchkonia T, Giorgadze N, Pirtskhalava T, et al. Fat depot origin affects adipogenesis in primary cultured and cloned human
Z. Alharbi et al.
23.
24. 25.
26.
27.
28.
29.
preadipocytes. Am J Physiol Regul Integr Comp Physiol 2002; 282:R1286e96. Oedayrajsingh-Varma MJ, van Ham SM, Knippenberg M, et al. Adipose tissue-derived mesenchymal stem cell yield and growth characteristics are affected by the tissue-harvesting procedure. Cytotherapy 2006;8:166e77. Coleman SR. Structural fat grafts: the ideal filler? Clin Plast Surg 2001;28:111e9. Fields RD, Lancaster MV. Dual-attribute continuous monitoring of cell proliferation/cytotoxicity. Am Biotechnol Lab 1993;11: 48e50. Kaufman MR, Bradley JP, Dickinson B, et al. Autologous fat transfer national consensus survey: trends in techniques for harvest, preparation, and application, and perception of shortand long-term results. Plast Reconstr Surg 2007;119:323e31. Ozsoy Z, Kul Z, Bilir A. The role of cannula diameter in improved adipocyte viability: a quantitative analysis. Aesthet Surg J 2006;26:287e9. Mojallal A, Lequeux C, Shipkov C, et al. Improvement of skin quality after fat grafting: clinical observation and an animal study. Plast Reconstr Surg 2009;124:765e74. Nguyen PS, Desouches C, Gay AM, Hautier A, Magalon G. Development of micro-injection as an innovative autologous fat graft technique: the use of adipose tissue as dermal filler. J Plast Reconstr Aesthet Surg 2012;65:1692e9.
Conventional vs. micro-fat harvesting: How fat harvesting technique affects tissue-engineering approaches using adipose tissue-derived stem/stromal cells ¨nder a,d, Sultan Almakadi a, Ziyad Alharbi a,b,*,d, Christian Opla Andrea Fritz a, Michael Vogt c, Norbert Pallua a Department of Plastic, Reconstructive and Hand Surgery e Burn Center, Medical Faculty, RWTH Aachen University, Pauwelsstr. 30, D-52074 Aachen, Germany b Division of Plastic and Reconstructive Surgery, Department of Surgery, King Abdullah Medical City, Kingdom of Saudi Arabia c Two-Photon Imaging Core Facility, Interdisciplinary Center for Clinical Research (IZKF), Medical Faculty, RWTH Aachen University, Pauwelsstr. 30, D-52074 Aachen, Germany a
Received 11 November 2012; accepted 10 April 2013
KEYWORDS Adipose tissuederived stem/stromal cells; Stromal vascular fraction; Liposuction; Lipofilling; Fat grafting; Biomaterials; Collagen-based scaffolds
Summary Background: Biocompatible scaffolds as dermal substitutes are used commonly in soft tissue reconstruction and tissue-engineering approaches. The combination of these scaffolds with mesenchymal stem and stromal cells would have additional benefits in multilayer soft tissue reconstruction. In addition, the use of lipoaspirate may be beneficial for this purpose containing high levels of regenerative cells and relevant growth factors. However there are many factors, which may impact the lipoaspirate content of isolated cells, cell behaviour and growth factors. There is a lack of data as to whether fat-harvesting procedures using different cannulas of small diameter will impact these parameters, which are relevant not only for tissue engineering but also for clinical outcome. Methods: Abdominal liposuctions were performed on 10 patients using the conventional fat harvesting by the Coleman cannula (3 mm, one-hole blunt tip) and the micro-fat-harvesting technique by the st’RIM cannula (2 mm, multi-perforated hole blunt tip) on contralateral area. Lipoaspirate contents of insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) were measured by enzyme-linked immunosorbent assay (ELISA). The in vitro viability of
* Corresponding author. Department of Plastic, Reconstructive and Hand Surgery e Burns Unit University Hospital of the RWTH Aachen University, Pauwelsstr. 30, 52057 Aachen, Germany. Tel.: þ49 241 80 0; fax: þ49 241 80 82634. E-mail address: [email protected] (Z. Alharbi). d Both authors contributed equally to this work. 1748-6815/$ - see front matter ª 2013 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bjps.2013.04.015
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Z. Alharbi et al. lipoaspirates was tested by the alamarBlue assay. Adipose-derived stem/stromal cells (ASCs) were isolated and the yields determined. Furthermore, ACSs were seeded on collagen elastin matrices (Matriderm) and cell migration/adhesion rate was examined by the alamarBlue assay and visualised by two-photon microscopy. Results: Conventionally obtained lipoaspirates were found to contain significantly higher concentrations of IGF and VEGF, but not PDGF or bFGF. No significant effects on the yields of ASCs or the in vitro viability of lipoaspirates obtained from different cannula sizes were observable. However, the viability and migration of isolated ASCs obtained from micro-harvested lipoaspirates were significantly higher. Moreover, a significant high adherence rate of isolated ASCs from the micro-fat-harvesting technique onto matrices was observed. Conclusion: The different sizes and surface/volume ratios of pieces of fatty tissue obtained by using different cannula sizes may be responsible for the observed differences and effects. Thus, micro-fat harvesting may be more suitable for tissue-engineering and -regenerative approaches using ASCs and collagen elastin matrices. ª 2013 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved.
Soft tissue augmentation by lipofilling with adipose tissue derived from liposuction procedures has shown promising results with less invasive techniques, but variation in longterm outcome in terms of loss of transplanted volume has been reported.1e3 One reason for a decreasing volume is insufficient blood supply after transplantation. Therefore, angiogenesis process is crucial for the survival of adipocytes, which are subject to apoptosis and necrosis if not supported by a network of capillary vessels by the fourth day after implantation.4 Studies have shown that the plasmatic nutritional supply of adipocytes can only go as far as 1.5 mm into tissues5 and that only 40% of the fat at a distance of 1.5 mm from the boundary of the tissue block survives.6 Growth factors, in particular, vascular endothelial growth factor (VEGF), are essential for angiogenesis, and after a short latency period the sprouting vessels organise into a capillary blood supply network, enabling adipocytes to survive.7 Lipoaspirates, obtained from liposuction, contain VEGF and many other growth factors, including basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF) and platelet-derived growth factor (PDGF).8 In animal studies, these growth factors have been shown to improve transplantation results.9e12 In the adipocyte-free fraction of lipoaspirate, the stromal vascular fraction (SVF) contains a variety of cells such as pre-adipocytes, endothelial cells, smooth muscle cells, pericytes, fibroblasts, macrophages and adult stem cells.13 Under certain conditions, these cells, called adipose-derived stem/stromal cells (ASCs), can be induced to differentiate into not only adipocytes but also bone, neuronal or endothelial cells. Therefore, ASCs, and especially the fraction of stem and progenitor cells, present an innovative option for regenerative cell therapy.14,15 For example, ASCs have been shown to play a major role in promoting wound healing16,17 and secrete almost all growth factors involved in normal wound healing.18 Furthermore, it has been reported that ASCs promote vessel density, the granulation process and collagen thickness16 and thus may improve the cosmetic aspect of the resultant scar.18 Since fatty tissue is a major source of stem cells19 and growth factors, the use of fatty tissue may provide more clinical benefits than augmentation alone.
However, studies have shown that apart from sex and depot-dependent differences,20e22 the yield of stem cells isolated from lipoaspirates can be affected by the fatharvesting procedure and/or lipofilling techniques.23 Furthermore, the combination of isolated adipose tissue derived cells with collagen-based scaffolds would provide another dimension in soft tissue reconstruction through tissue-engineering approaches. Clinically, collagen elastin matrices (Matriderm) have been used in multistage operations or even in one-stage operations as they have been cooperated with autologous split thickness skin transfer for soft tissue reconstruction. Variables such as the ideal cannula and technique for harvesting, the best way of processing fat to ensure maximum uptake and viability of the graft and ACS on collagen elastin matrices, are all factors which require clarification.2 For using fresh-isolated ACSs for tissueengineering or -regenerative approaches the harvesting technique may be crucial, because cells have to adhere fast onto scaffolds to avoid ‘cell slipping’ away from scaffold during transplantation or from the original site of recipient area after transplantation. Also factors, such as cell yield, viability and growth factors, influenced by the harvesting technique may affect the performance of ACS-based tissue engineering for regenerative medicine purposes. Therefore, the aim of this study is to compare the influence of the fat-harvesting procedures (conventional vs. micro-fatharvesting technique) on the viability and content of growth factors of the grafts obtained. Furthermore, we have investigated the effects of both methods on the yield, migration and adhesion rate of the ASCs which have been isolated directly from the corresponding lipoaspirates.
Methods and materials Patients With the patients’ consents, lipoaspirates were obtained from 10 healthy patients (five males and five females; age range 27e59) having undergone an elective liposuction procedure
Conventional vs. micro-fat harvesting from a single anatomical site (abdominal subcutaneous fat tissue) at the Department of Plastic and Hand Surgery e Burns Centre, RWTH Aachen University Hospital. Fat tissues have been obtained from the deep layer by the same surgeon (NP). Patients with body mass index (BMI) >30, diabetes, hypertension and nicotine or alcoholic abuse have been excluded. The protocol and use of lipoaspirates were approved by the regional ethics committee (EK163/07) and conducted in compliance with the Declaration of Helsinki Principles.
Liposuction procedures Conventional fat-harvesting technique This established method was performed as described by SR Coleman.24 A mixed solution (1 liter of sodium chloride 0.9%, and 12.5 ml of prilocaine 1%, and 1 ml of epinephrine 1:200,000) was infiltrated into the donor site. The solution was infiltrated at a ratio of 1 ml of solution per cubic centimetre. The fat grafts were harvested through the same incisions. The harvesting cannula was 3 mm in diameter with a single-hole blunt tip (Byron Medical Inc., Tucson, AZ, USA) connected to a 10-ml syringe (see Figure 1). After filling the syringe with harvested tissue (20 cc), the cannula was removed from the syringe. Micro-fat-harvesting technique The procedure as outlined above was repeated in the contralateral region to obtain another 20 cc of harvested fat. However, in contrast to the Coleman cannula, the used harvesting cannula was an st’RIM cannula (Thiebaud Biomedical Devices, Margencel, France) developed by Guy Magalon for micro-lipografting. This cannula was 2 mm in diameter with a blunt tip and four 600-mm-gauged orifices (see Figure 1) (for details, see http://www.mystrim.com/surgeon/).
Washing and centrifugation of the obtained lipoaspirate With each patient, the two different liposuction techniques were performed. Immediately after the liposuction procedure, the body of the filled syringe was placed into a centrifuge (Sigma 2e16 K, Osterode am Harz, Germany) and centrifuged (3 min, 300 rpm). After centrifugation, the oil layer (upper level) was decanted and the aqueous layer (lower level) was drained from the syringe. The middle layer, composed of predominantly fat grafts, represents the purified lipoaspirate, which was used immediately for experiments and for cell isolations.
Isolation of SVF cells from lipoaspirate From each patient with special consideration to the selected fat-harvesting procedure, a volume of 20 ml of purified lipoaspirate, from each method, was transferred into a sterile
Figure 1 Shows the 2 mm st’Rim harvesting cannula (above) and the 3 mm Coleman harvesting cannula blunt type (below).
1273 tube and 30 ml of normal saline was added. Then, a second centrifugation process was completed for 10 min at 300 rpm. After centrifugation, adipose tissue was transferred into a sterile tube for the isolation process. Collagenase solution was freshly prepared (1% bovine serum albumin (PAA, Linz, Austria) and 2% collagenase type 1 (Biochrom, Berlin, Germany)) in a buffer solution (100 mM 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES), 120 mM NaCl, 50 mM KCl, 1 mM CaCl2, 5 mM glucose, pH 7.4) and then was added to the centrifuged adipose tissue through a 0.2-mm filter under sterile conditions (enzyme/tissue ratio is 1:1). After incubation (45 min, 37 C) and under constant shaking, the digested tissue solution was filtered using a 250-mm filter (Neolab, Heidelberg, Germany) and centrifuged for 10 min at 300 g. Yellow rigid fatty tissue and supernatant were discarded and the pellet was resuspended with 30 ml NaCl solution and then was re-centrifuged at 300 g for another 10 min. The pellet was resuspended in DMEM/F12 cell culture medium (Invitrogen, Darmstadt, Germany) supplemented with 1% penicillin and 1% streptomycin (PAA, Linz, Austria) without further proliferation factors.
Determination of the content of isolated ASCs Isolated cells were counted manually under a light microscope using a Neubauer chamber (VWR, Darmstadt, Germany). A trypan blue assay made it possible to exclude dead cells. Then 10 mm trypan blue (PAA, Linz, Austria) was added to 10 ml of cell suspension before counting. Experiments using ASCs started directly after isolation.
Testing fat tissue viability and survival rate in vitro In parallel, purified lipoaspirates obtained from conventional fat-harvesting or micro-fat-harvesting procedures were transferred into a six-cell culture plate (1 g for each well) and 2 ml of DMEM/F12 cell culture medium supplemented with 1% penicillin/1% streptomycin/10% foetal calf serum added to each well. For testing the viability of lipoaspirates within the first 24 h after liposuction, 300 ml of alamarBlue reagent (AbD Serotec, Oxford, UK) was added into some wells and after 24 h incubation time (5% CO2, 37 C). The 2 100 ml Medium/alamarBlue mix was carefully removed from the well and immediately measured at room temperature using a fluorescence spectrometer (Fluostar Optima; BMG Labtech, Offenburg, Germany; excitation wavelength 540 nm, emission wavelength 590 nm).25 For testing the viability of lipoaspirates at 24 and 48 h after liposuction, 300 ml alamarBlue reagent was now added to the other wells and incubated for further 24 h (5% CO2, 37 C). After incubation, 2 100 ml Medium/alamarBlue mix was carefully removed from the well for fluorescence measurement.
Measurement of growth factors and cytokines in lipoaspirate fat tissue Purified lipoaspirate (1 g) was frozen immediately at 80 C. To evaluate the content of growth factors by enzyme-linked immunosorbent assay (ELISA), the samples were thawed on ice at 4 C, and 3 ml of homogenisation buffer (pH 7.5) was added to each sample. An Ultra-Turrax
1274 homogeniser (IKA Works, Inc., Wilmington, NC, USA) was applied twice for 30 s and the homogenised suspension was centrifuged (100 g, 10 min, 4 C). The supernatant was again centrifuged (20,000 g, 40 min, 4 C) and the oily phase and cell pellet were discarded. The supernatant obtained was divided into aliquots, shock-frozen in liquid nitrogen and stored at 80 C. To measure the content of bFGF, IGF-1, VEGF and PDGF-BB in the centrifuged lipoaspirate obtained from the conventional fat-harvesting technique and micro-fat-harvesting procedures, sample aliquots were analysed using appropriate ELISA duo sets (ELISA, R&D Systems, Minneapolis, MN, USA). Extinction was measured as recommended by the manufacturer using a FLUOstar OPTIMA microplate reader at 450 nm, and by using a benchmark value of 540 nm.
Incubation of Matriderm with ASCs or purified lipoaspirate to determine cell migration and adherence rate Matriderm sheets (1 mm thick) consisting of non-crosslinked native bovine collagen matrix (type I, III and V collagen derived from bovine skin) were used in this study (supplied by Dr. Otto Suwelack Skin and Health Care GmbH, Billerbeck, Germany). Circular pieces of Matriderm were obtained using a circular punch biopsy device measuring 0.8 cm, and then placed on 46-well culture plates. Custommade sterile tubes (diameter 0.8 cm) were used to fix the sheets and to avoid direct contact between cells and the bottom of the cell culture wells. Fat graft (1 g) was directly transferred into 46-well cell culture plates lined with Matriderm and incubated at 37 C and 5% CO2 for 24 h. Alternatively ASCs were directly added at a density of 5 104 (270 ml DMEM/F12) into 48-well culture plates lined with Matriderm and incubated at 37 C and 5% CO2 for 24 h in DMEM/F12 cell culture medium supplemented with 1% penicillin and 1% streptomycin with no proliferation factors.
Z. Alharbi et al. Japan, attached to a pulsed Ti-Sapphire laser from MaiTai DeepSee, SpectraPhysics). The collagen elastin matrix was visualized by the non-linear optical effect of second harmonic generation (SHG). For excitation of FDA and SHG the laser was tuned to the wavelength of 850 nm. The emission of FDA and SHG was collected at 495-540 nm and 418-468 nm respectively. Consecutively Hoechst was excited at 730 nm and detected at 418-468 nm. Series of subsequent xy-frames with 1024 1024 pixels in 1 mm z-steps were obtained for structural 3D reconstruction conducted with Imaris Software (Bitplane, Zurich, Switzerland).
Results Comparison of cell yields successfully isolated from conventional and micro-fat-harvesting techniques From lipoaspirates gained from either conventional or micro-fat-harvesting techniques, ASCs were isolated and
Determination of the number of isolated cells adhered at the matrix The number of viable ASCs was tested by using the alamarBlue assay. The Matriderm sheet, incubated with purified lipoaspirate or isolated ASCs, was separated after the 24-h incubation period. The sheet was washed carefully with normal saline (0.9% NaCl) and transferred into a clean culture plate. After suspension using 270 ml of pre-adipocyte medium (DMEM/F12 supplemented with 100 U ml1 penicillin and 100 mg ml1 streptomycin) with no proliferation factors, 30 ml of alamarBlue reagent was added to each well. After incubation for 2 h (37 C, 5% CO2), samples were measured directly at room temperature by a fluorescence spectrometer (excitation wavelength 540 nm, emission wavelength 590 nm).
Two-photon microscopy For vital staining of the isolated cells and visualisation of the adherent cells on the Matriderm, fluorescein diacetate (FDA) and Hoechst 33342 were added. The evaluation was carried out in just 24 h after seeding of the ASCs using a twophoton microscope (FV1000MPE, Olympus Corp., Tokyo,
Figure 2 A shows the quantification of the numbers of cells (ASCs) which could be isolated from lipoaspirates obtained by conventional and by micro-fat harvesting technique (20 g) under standard isolation procedure. B shows the concentration of Basic Fibroblast Growth Factor (bFGF), Insulin-like Growth Factor 1 (IGF-1), Vascular Endothelial Growth Factor (VEGF) and Platelet-derived Growth Factor (PDGF) found in homogenates of purified lipoaspirates obtained by by conventional and by microfat harvesting determined by ELISA; n Z 10; *p < 0.05.
Conventional vs. micro-fat harvesting
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yields were determined. Mean values are shown in Figure 2A. There were no significant differences in cell yields between the two techniques and standard deviations were in the same range, indicating strong intra-individual variations. However, the mean value for the micro-fatharvesting technique (3.60 107 cells g1 lipoaspirate) appears somewhat greater than the value from the conventional technique (2.94 107 cells g1 lipoaspirate).
Determination of growth factors of purified lipoaspirates Purified lipoaspirates from either the conventional or micro-fat-harvesting technique were homogenised and the concentration of growth factors relevant for cell growth and angiogenesis, such as bFGF, IGF-1, VEGF and PDGF, was determined by ELISA. As shown in Figure 2B, the concentrations of IGF-1 and VEGF found in both types of lipoaspirates were significantly different. The IGF-1 concentration of conventional lipoaspirates was about twice as great as the concentration of lipoaspirates obtained from the micro-fat-harvesting technique (192 128 vs. 77 83 pg mg1). Furthermore, significant differences were found in the VEGF concentrations, where conventional lipoaspirates were found to contain 52 29 pg mg1 against a figure of only 35 18 pg mg1 for the lipoaspirates obtained from the micro-fat-harvesting procedure. No significant differences were found in relation to bFGF and PDGF-BB concentrations.
Comparison of in vitro vitality of conventional and micro-fat grafts By using the alamarBlue assay, it was possible to carry out an estimation of living cells and fat graft vitality in vitro. After 24 h, the alamarBlue fluorescence values indicate the presence of living cells in the fat grafts. In our investigation, the mean signal relating to fat grafts obtained from the conventional technique was slightly, but not significantly, higher than the grafts obtained from the micro-fat-harvesting technique (Figure 3A). After 48 h, a significant decrease in fluorescence values was observed for both techniques, indicating a loss of cell vitality (38% conventional vs. 26% mico-lipografting) in the fat grafts.
Determination of lipoaspirate cell/ASC migration and adherence onto collagen matrices To determine the cell migration from lipoaspirates onto a collagen matrix, Matriderm sheets were incubated with lipoaspirates obtained from conventional and micro-fatharvesting techniques respectively, and the relative cell number of adherent cells was measured by the alamarBlue assay after 24 h. As shown in Figure 3B, significant, twice as high alamarBlue fluorescence signals were observed on Matriderm sheets incubated with lipoaspirates obtained by the micro-fat-harvesting technique (4311 1553 AU) than those obtained by the conventional technique (2071 747 AU). As shown in Figure 3C, with fluorescence values normalised to the cell number determined in the lipoaspirate in each instance and the
Figure 3 A shows the in vitro vitality of purified lipoaspirates obtained by conventional and by micro-fat harvesting in 24 h and 48 h after liposuction, B shows the number of cells adhering to the Matriderm collagen sheets after 24 h incubation with purified lipoaspirates. C shows values normalised to the cell number determined in the used lipoaspirate in particular. Mean and standard deviation from 6 to 10 independent experiments are provided; *p < 0.05.
difference between the numbers of translocated cells remains significant (micro-lipografting 11.95 6.69 AU vs. conventional lipografting 7.13 2.87 AU normalised to 50,000 cells), indicating that the greater cell number found in micro-fat-harvesting procedure is not the only reason for the higher migration and adherence rate of cells.
1276 Furthermore, there is also a significant difference in the adhesion rate between freshly isolated ASCs from lipoaspirates obtained by the two different techniques. Here, as shown in Figure 4A, Matriderm sheets incubated for 24 h with a defined number of cells isolated from the micro-fatharvesting technique showed significantly higher fluorescence values than those obtained by the conventional technique (6537 3667 AU vs. 2892 1547 AU). Moreover, the exemplary visualisation of the Matriderm sheets using two-photon microscopy shows that the seeded isolated adipose-derived cells could be found within the Matriderm collagen matrix after a 24-h incubation time (Figure 4B).
Discussion Today, autologous fat grafting is a beneficial procedure for patients with volume loss in reconstructive and cosmetic
Figure 4 A shows the alamarBlue fluorescence values, which are proportional to number of viable cells on Matriderm sheets incubated for 24 h with isolated ASCs obtained by conventional and micro-fat harvesting technique. Mean and standard deviation of 6 independent experiments are provided; *p < 0.05. B shows exemplary 3-D visualisation of ASCs which were adherent after 24 h incubation of the Matriderm sheet with isolated ASCs.
Z. Alharbi et al. surgery. However, the resorption rate is seen to be a variable and the loss of volume may require further grafting procedures.26 One point for consideration is the method of ¨ zsoy et al. stated that the use of widerliposuction. O diameter cannulas can potentially improve fat graft survival and reduce fat graft resorption.27 One important characteristic of adipose tissue and adipose-derived stem/ stromal cells is the secretion of a variety of growth factors and cytokines,8 which can be beneficial for patients. For example, using nude mice as a model, Mojallal et al. found a local increase of type I collagen fibres of murine origin after injection of human fatty tissue resulting from murine fibroblast stimulation by the human fatty tissue. Additionally, dermal thickness significantly increased around the injection area.28 In this study, we used the conventional Coleman technique (3-mm-diameter blunt-type cannula) and the micro-fat-harvesting technique (2-mm-diameter blunt-type cannula with multiple perforated orifices of 600 mmgauged) on the same patients in the abdominal region only with the same centrifuge power and time, thus allowing us to focus on the impact of cannula size on graft viability, ACS yield, growth factor concentrations and cell behaviour. We observed slight, but not significant, differences in the yields of ASCs (Figure 2). Under application of the same isolation procedure, lipoaspirates obtained by the micro-fatharvesting technique yielded cell numbers 22.4% higher than obtained with the conventional technique. However, differences in cell numbers between patients were high, resulting in high standard deviations. Significantly, higher VEGF and IGF-1 concentrations (but not bFGF and PDGF-BB) were found in conventional obtained lipoaspirates. By using a smaller cannula size (st’RIM) with small 600-mm-gauged orifices, lipoaspirates with smaller tissue pieces are obtained, where the surface/volume ratio is higher compared with larger tissue pieces obtained by a larger cannula and size (Coleman technique), leading to an increased loss of secreted soluble of VEGF and IGF-1 during the washing steps of lipoaspirate purification. Since the development of bFGF and PDGF-BB is more intracellular, their total concentrations would be impacted by the cell numbers in the pieces of tissue rather than by the surface/volume ratio and washing steps, which in our case are not significantly different. A significant loss of vitality after 48 h was identified in conventional lipoaspirates. However there was also a loss of vitality in the case of the micro-fat-harvesting lipoaspirates, indicating that it was not significant. A possible reason for this difference may be a reduced oxygen supply via diffusion for the larger pieces of tissue of the grafts obtained by conventional liposuction. However, a recent study from Nguyen et al. in 2012 showed that following fat transplantation into mice, the viability and volume of fat grafts obtained using a classical 3-mm Coleman harvesting cannula were not significantly different in comparison to fat grafts obtained by multiperforated cannula with holes of 600 mm-gauged in diameter.29 Furthermore, both injected fat grafts maintained a normal histological structure. Apart from clinical aspects, for tissue-engineering and -regenerative approaches the yields, viability and cell behaviour of ASCs freshly isolated from lipoaspirates can be impacted by many parameters, such as harvesting procedure.23 In such cases, for optimal results, the ideal cannula
Conventional vs. micro-fat harvesting and technique for harvesting, and also the best way and time of processing the fat graft for cell isolation have yet to be identified.2 In our study we could observe that the harvesting technique influences the ASC migration significantly. Here, ASCs migrate more likely form micro-fat grafts to a collageneelastin matrix (Matriderm) than conventional grafts. This may be important for enriching scaffolds with ASCs for tissue engineering without further processing steps for cell isolation and culturing. The higher surface/volume ratio of fatty pieces of tissue in lipoaspirates obtained from the micro-fat-harvesting technique could explain the results observed. In an in vivo situation, a higher migration rate from cells of the graft to the existing intracellular matrix in the recipient area may provide beneficial effects for the patients. In addition, the finer lipoaspirates obtained by micro-fat harvesting allow an application onto the superficial layer of the skin using smaller cannulas, which would be clogged by conventional lipoaspirates. Interestingly, after cell isolation, the adhesion rate of ASCs from micro-fat harvesting to the collagen matrix is twice as high as that of ASCs obtained from the conventional liposuction technique. This may be explained by differences in cell populations obtained after isolation. Moreover, the observed differences in the concentration of growth factors in lipoaspirates may impact the adhesion response of ASCs. To recapitulate, the study has shown that the technique and the cannula used for liposuction can impact the number of cells and the viability of the lipoaspirate tissue. Furthermore, the liposuction technique used has a significant impact on growth factor concentrations of purified lipoaspirates, cell migration from pieces of fatty tissue and also the adherence rate of ASCs. The probability is that these observed effects are due to the different sizes and surface/volume ratios of pieces of fatty tissue obtained using different cannula sizes. Such results are important not merely for a scientist in the bench side but also for clinicians who want to drive more viable isolated cells into a clinical tissue-engineering approach for multilayer soft tissue reconstruction in an autologous approach. We advance the hypothesis that a minimal fat lobule size not only is required for graft survival after lipofilling but also may represent an important factor for adipose-derived stem cell-based approaches. Nevertheless, further experimental studies and clinical trials have to be conducted to support such an assumption.
Conclusion The different sizes and surface/volume ratios of pieces of fatty tissue obtained by using different cannula sizes may be responsible for the observed differences and effects. Thus, micro-fat harvesting may be more suitable for tissueengineering and -regenerative approaches using ASCs and collageneelastin matrices.
Declaration The use of human material was approved by the local ethics committee of the Medical Faculty of the RWTH University
1277 Aachen (Number: EK163/07) and conducted in compliance with the Declaration of Helsinki Principles.
Disclosures Authors state no disclosures.
Conflict of interest The authors state no conflict of interest.
Acknowledgements We thank Dr. Otto Suwelack Skin and Health Care GmbH for supplying the Matriderm sheets. This work was partially supported by the Two-Photon Imaging Core Facility for imaging process, a core facility of the Interdisciplinary Center for Clinical Research (IZKF) Aachen within the Faculty of Medicine at RWTH Aachen University.
References 1. Boschert MT, Beckert BW, Puckett CL, Concannon MJ. Analysis of lipocyte viability after liposuction. Plast Reconstr Surg 2002; 109:761e5 [discussion 66e7]. 2. Bucky LP, Percec I. The science of autologous fat grafting: views on current and future approaches to neoadipogenesis. Aesthet Surg J 2008;28:313e21 [quiz 22e4]. 3. Sommer B, Sattler G. Current concepts of fat graft survival: histology of aspirated adipose tissue and review of the literature. Dermatol Surg 2000;26:1159e66. 4. Crandall DL, Hausman GJ, Kral JG. A review of the microcirculation of adipose tissue: anatomic, metabolic, and angiogenic perspectives. Microcirculation 1997;4:211e32. 5. Cortese A, Savastano G, Felicetta L. Free fat transplantation for facial tissue augmentation. J Oral Maxillofac Surg 2000;58: 164e9 [discussion 69e70]. 6. Carpaneda CA, Ribeiro MT. Percentage of graft viability versus injected volume in adipose autotransplants. Aesthetic Plast Surg 1994;18:17e9. 7. Yamaguchi M, Matsumoto F, Bujo H, et al. Revascularization determines volume retention and gene expression by fat grafts in mice. Exp Biol Med (Maywood) 2005;230:742e8. 8. Pallua N, Pulsfort AK, Suschek C, Wolter TP. Content of the growth factors bFGF, IGF-1, VEGF, and PDGF-BB in freshly harvested lipoaspirate after centrifugation and incubation. Plast Reconstr Surg 2009;123:826e33. 9. Schweigerer L. Fibroblast growth factor and angiogenesis. Z Kardiol 1989;78(Suppl. 6):12e5. 10. Eppley BL, Sadove AM. A physicochemical approach to improving free fat graft survival: preliminary observations. Aesthetic Plast Surg 1991;15:215e8. 11. Bluher S, Kratzsch J, Kiess W. Insulin-like growth factor I, growth hormone and insulin in white adipose tissue. Best Pract Res Clin Endocrinol Metab 2005;19:577e87. 12. Hausman GJ, Richardson RL. Adipose tissue angiogenesis. J Anim Sci 2004;82:925e34. 13. Witkowska-Zimny M, Walenko K. Stem cells from adipose tissue. Cell Mol Biol Lett 2011;16:236e57. 14. Mizuno H, Tobita M, Uysal AC. Concise review: adipose-derived stem cells as a novel tool for future regenerative medicine. Stem Cells;30:804e10.
1278 15. Gimble JM, Bunnell BA, Chiu ES, Guilak F. Concise review: adipose-derived stromal vascular fraction cells and stem cells: let’s not get lost in translation. Stem Cells;29: 749e54. 16. Ebrahimian TG, Pouzoulet F, Squiban C, et al. Cell therapy based on adipose tissue-derived stromal cells promotes physiological and pathological wound healing. Arterioscler Thromb Vasc Biol 2009;29:503e10. 17. Tobita M, Mizuno H. Adipose-derived stem cells for periodontal tissue regeneration. Methods Mol Biol;702:461e70. 18. Blanton MW, Hadad I, Johnstone BH, et al. Adipose stromal cells and platelet-rich plasma therapies synergistically increase revascularization during wound healing. Plast Reconstr Surg 2009;123:56Se64S. 19. Zuk PA, Zhu M, Ashjian P, et al. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell 2002;13: 4279e95. 20. Padoin AV, Braga-Silva J, Martins P, et al. Sources of processed lipoaspirate cells: influence of donor site on cell concentration. Plast Reconstr Surg 2008;122:614e8. 21. Fraser JK, Wulur I, Alfonso Z, Zhu M, Wheeler ES. Differences in stem and progenitor cell yield in different subcutaneous adipose tissue depots. Cytotherapy 2007;9:459e67. 22. Tchkonia T, Giorgadze N, Pirtskhalava T, et al. Fat depot origin affects adipogenesis in primary cultured and cloned human
Z. Alharbi et al.
23.
24. 25.
26.
27.
28.
29.
preadipocytes. Am J Physiol Regul Integr Comp Physiol 2002; 282:R1286e96. Oedayrajsingh-Varma MJ, van Ham SM, Knippenberg M, et al. Adipose tissue-derived mesenchymal stem cell yield and growth characteristics are affected by the tissue-harvesting procedure. Cytotherapy 2006;8:166e77. Coleman SR. Structural fat grafts: the ideal filler? Clin Plast Surg 2001;28:111e9. Fields RD, Lancaster MV. Dual-attribute continuous monitoring of cell proliferation/cytotoxicity. Am Biotechnol Lab 1993;11: 48e50. Kaufman MR, Bradley JP, Dickinson B, et al. Autologous fat transfer national consensus survey: trends in techniques for harvest, preparation, and application, and perception of shortand long-term results. Plast Reconstr Surg 2007;119:323e31. Ozsoy Z, Kul Z, Bilir A. The role of cannula diameter in improved adipocyte viability: a quantitative analysis. Aesthet Surg J 2006;26:287e9. Mojallal A, Lequeux C, Shipkov C, et al. Improvement of skin quality after fat grafting: clinical observation and an animal study. Plast Reconstr Surg 2009;124:765e74. Nguyen PS, Desouches C, Gay AM, Hautier A, Magalon G. Development of micro-injection as an innovative autologous fat graft technique: the use of adipose tissue as dermal filler. J Plast Reconstr Aesthet Surg 2012;65:1692e9.