RESEARCH
Effect of Calcium Alginate Microsphere Loaded With Vascular Endothelial Growth Factor on Adipose Tissue Transplantation Shi-Li Ding, MD, Meng-Yuan Zhang, MD, Song-Jia Tang, MD, Hu Yang, MD, and Wei-Qiang Tan, MD Abstract: Revascularization in the early period after transplantation is the key to improving adipocyte survival. Vascular endothelial growth factor (VEGF) is known as the master regulator of angiogenesis. However, consensus is lacking regarding safe and efficient methods for applying VEGF in free fat transplantation in the clinical setting. We constructed calcium alginate (CA) microspheres loaded with VEGF to increase the survival of implanted adipocytes. BALB/c nude mice were used as adipose tissue transplantation receptors. Adipocytes were mixed with CA microspheres loaded with VEGF and implanted subcutaneously into the dorsum of mice. Grafts were harvested at week 3, 6, and 12 after transplantation. We found that the mass and microvascular density of grafts in the VEGF + CA group (CA microspheres loaded with VEGF) were statistically higher than that of other groups in a timedependent manner. We demonstrated that CA microspheres loaded with VEGF can significantly promote the fat graft neovascularization, thus improving adipocyte survival. Key Words: revascularization, vascular endothelial growth factor, calcium alginate, free fat transplantation (Ann Plast Surg 2015;75: 644Y651)
A
utologous adipose tissue is usually considered an ideal filler material in plastic surgery because it is versatile, natural-appearing, inexpensive, biocompatible, and available in a sufficient amount.1Y4 Furthermore, the application of injection method gives autologous adipose tissue transplantation the advantages of simple manipulation and minimal trauma. Clinically, this technique has been widely used in correcting surface depression or augmenting soft tissue. However, with an absorbance of 25% to 80%,4Y8 the unpredictable and often low survival of the autologous fat tissue affects the outcome of this technique. In the past 20 years, many studies have been done to increase the viability of autogenous fat tissue.9Y18 The view that a fast blood supply reestablishment is the key to improving adipocyte survival has been widely accepted. Thereby, vascular endothelial growth factor (VEGF) became the research hot topic.13,19,20 Vascular endothelial growth factor is the master regulator of angiogenesis both in physical and pathological development.21 It is Received August 29, 2013, and accepted for publication, after revision, February 12, 2014. From the Department of Plastic Surgery, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, People’s Republic of China. Dr Ding and Dr Zhang contributed equally to this article as first authors. Conflicts of interest and sources of funding: This work was supported by funding from the National Natural Science Foundation of China (No. 81372072 and 30800228), Zhejiang Provincial Medical and Healthy Science Foundation of China (No. 2013RCB004), and Zhejiang Provincial Natural Science Foundation of China (No. LY12H15006). Reprints: Wei Qiang Tan, MD, Department of Plastic Surgery, The First Affiliated Hospital, College of Medicine, Zhejiang University, 79 Qingchun Rd, Hangzhou, Zhejiang Province 310003, People’s Republic of China. E-mail: [email protected]. Copyright * 2014 Wolters Kluwer Health, Inc. All rights reserved. ISSN: 0148-7043/15/7506-0644 DOI: 10.1097/SAP.0000000000000201
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capable of starting the complex cascade of events leading to endothelial cell activation, assembly of new vascular structures, mural cell recruitment, and vessel stabilization.22 However, the incorporation of growth factors involves many difficulties because of their short half-lives of only several minutes in circulation.23,24 Free VEGF can barely exert its biological function when applied directly25 because it tends to diffuse, dilute, and degrade, resulting in very low bioavailability. We applied calcium alginate (CA) as drug carrier to achieve a controlled release of VEGF. Alginates are a family of natural anionic and biocompatible polysaccharides derived from brown algae. They are very suitable for encapsulating biomacromolecules and living cells because of their low toxicity, high biocompatibility, and timely degradation.26Y28 We constructed CA microspheres loaded with VEGF to improve the bioavailability of VEGF. In addition, we mixed them with implanted adipocytes to increase adipocyte survival rate. In our animal experiment, the mixture was injected subcutaneously into the dorsa of nude mice. The in vivo angiogenic response to the controlledrelease system was measured by evaluating the microvessel density (MVD) with immunohistochemistry.
MATERIALS AND METHODS The animal experiment designed in this study was approved by the Ethics Committee of Zhejiang University.
Preparation of CA Microspheres Loaded With VEGF A solution of 1.2% sodium alginate (wt/vol; Sigma, San Diego, Calif ) in 0.9% (wt/vol) sodium chloride was magnetic stirred for 2 hours until completely dissolved. Then, the solution was sterilized by a high-pressure sterilizer for 40 minutes at 121-C. After that, it was dried and preserved at 4-C for later procedure. One milliliter of VEGF165 (1 Kg/mL; Peprotech, Rocky Hill, NJ) was added into 4 mL of sodium alginate solution to obtain 5 mL of VEGF solution (0.2 Kg/mL). Then, the dilution was slowly extruded dropwise through a 23-gauge hypodermic needle into 10 mL of calcium chloride solution (11.3 g/L). Wait for 10 minutes at room temperature to reach a good cross-linking effect. Then, the rest of the calcium chloride solution was removed, and the microspheres were rinsed for 5 times with 0.9% (wt/vol) sodium chloride (Fig. 1).
Adipose Tissue Transplantation A liposuction needle (2.5-mm caliber) was connected to a 20-mL syringe. Adipose tissue was suctioned subcutaneously from the abdomen of a healthy young female patient. Collagenase I (Sigma, St Louis, Mo) was dissolved with Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Carlsbad, NM) in a 37-C thermostatic water bath and filled into 16 centrifuge tubes, 4 mL each (1.0 g/L). Two milliliters of adipose tissue (32 mL in total) was filled into each centrifuge tube. The adipose tissue was digested for 1 hour. After that, each tube was filled Annals of Plastic Surgery
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FIGURE 3. The grafts were distributed well proportioned to avoid merging.
FIGURE 1. The CA microspheres can be seen attaching to the surface of the centrifuge tube.
with 6 mL of DMEM (supplemented with 10% fetal bovine serum) to terminate the digestion. Then, the mixtures were centrifuged at 1200 rpm for 10 minutes. The mixtures appeared as 4 layers from top to bottom: lipid, adipocytes, supernatant, and pellet (Fig. 2). Adipocytes were carefully preserved for subsequent procedure. A total of 4.5 mL of adipocytes was mixed with 1.5 mL of the following materials before transplantation: CA microspheres loaded with VEGF (0.2 Kg/mL), CA microspheres, VEGF (0.2 Kg/mL), and DMEM. Six- to 8-week-old BALB/c nude mice weighing 20 to 25 g were selected regardless of sex (provided by the experimental animal center of Zhejiang University). Twenty-four mice were equally divided into 4 cages at random. The abovementioned adipose grafts were implanted subcutaneously into the dorsum of each mouse at 4 points with a sharp 12gauge needle attached to a 1-mL syringe. Each point was injected with 0.2 mL of the graft; the adipocyte amount was 3 105 per site. The grafts of different composition were assigned to the following groups: VEGF + CA group (adipocytes and CA microspheres loaded with VEGF), CA group (adipocytes and blank CA microspheres), VEGF group (adipocytes and free VEGF), and blank control group
(adipocytes only). The grafts were distributed well proportioned to avoid merging (Fig. 3). By this way, the 4 grafts in each mouse were set symmetrically, and their sites were rotated clockwise to eliminate potential skewing of results. Mice in the same cage shared the same order of injection sites.
Sample Collection and Histopathological Measurement Two mice in each cage were killed at weeks 3, 6, and 12 after transplantation. The fat grafts were carefully stripped out and weighed. The exterior characteristics of the graft were visually observed. Sections of grafts were observed under a light microscope (Eclipse 80i; Nikon) after staining with hematoxylin and eosin (HE). Observation of adipocytes, degree of inf lammation, and fibrosis were recorded. The grafts harvested at the third and sixth weeks of the VEGF + CA group were sent for scanning electron microscopy and transmission electron microscopy.
Microvascular Density Determination Samples were paraffin embedded and sliced into 4-Km sections. The expression of CD34 was detected by means of immunohistochemical staining. Microvessels among the adipocytes were observed under a light microscope (Eclipse 80i; Nikon) at low magnification (100) to find the area where the highest vascular density was located. Then, 4 fields of fixed magnification (400) were chosen randomly, and images were saved. The relative area of stained microvessel in each image was calculated by Image-Pro Plus 6.0 software (Media Cybernetics; Rockville, Md).
Statistical Analysis Data are presented as mean (SD). All data were analyzed with the Statistical Package for the Social Sciences 13.0 software (IBM, Chicago, Ill). Comparisons of means of several groups were tested with 1-way analysis of variance (ANOVA). Comparisons of survival between every 2 groups at different times were tested by least significant difference (LSD) method. Probability values of P G 0.05 were interpreted to denote statistical significance.
RESULTS All animals tolerated the operations well. There was no evidence of any ulceration, infection, hematoma, or seroma at the injection points.
General Observation of Adipose Grafts FIGURE 2. The mixture appeared as 4 layers from top to bottom: lipid, adipocytes, supernatant, and pellet. * 2014 Wolters Kluwer Health, Inc. All rights reserved
The grafts were yellow, soft, and easily dissectible. They also had well-defined periphery with intact envelope (Fig. 4). Vessels were visible from the grafts’ surfaces. There were more vessels on the www.annalsplasticsurgery.com
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FIGURE 4. A graft harvested from the dorsum of a mouse at the third week in the VEGF + CA group (upper left). The graft is made up of yellow adipose tissue and transparent CA microspheres. The other 3 pictures are the grafts harvested at weeks 3, 6, and 12. The grafts were yellow, soft, and easily dissectible. They also had well-defined periphery with intact envelope. Vessels were visible from the surface of grafts. There were more vessels on the surface of grafts of the VEGF + CA group than other groups. Not completely absorbed CA can be seen from the grafts that were harvested at the third week. Grafts in the VEGF + CA group have a larger volume than grafts in other groups in all periods.
surface of the grafts of the VEGF + CA group than other groups. Fat liquefaction happened among the grafts individually. Not completely absorbed CA can be seen from the grafts that were harvested at the third week.
Histological Evaluation At the third week, the grafts were surrounded by a thin envelope of fibrous tissue. Inf lammatory cellular infiltration was not found in or around the adipose tissue. Vascular invasion could be seen in all 4 groups. Focal necrosis in the VEGF + CA group was less common than in other groups. The form and the size of adipocytes in the VEGF + CA group were relatively uniform whereas varied in other groups (Fig. 5). At the sixth week, the number of vessels increased. Ingrowth of fibrous tissue could be seen in every group, especially in the VEGF group and the blank control group. Necrotic areas were even larger than 3 weeks ago (Fig. 6). In the grafts obtained at the 12th week, larger necrotic areas (with or without punctate calcification) and less severe fibrosis were observed in the VEGF + CA group than in other groups. There were more fat cells that survived in the VEGF + CA group, whereas only small clusters of fat cells survived in other groups (Fig. 7).
Electron Microscopy Observation There are CA microspheres surrounding the adipocytes in the sections of both the third week and the sixth week when observed with scanning electron microscope. The edges between cells were indistinct (Fig. 8). Calcium alginate microspheres can be obviously seen 646
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outside the adipocytes, viewed with transmission electron microscope. No incepted CA was noticed inside the adipocytes. No distinct fibrous tissue was noticed covering the surfaces of adipocytes or CA microspheres too (Fig. 9).
Graft Mass The graft masses of the 4 groups at weeks 3, 6, and 12 were shown in Table 1. Figure 10 showed that the mass decrease in the VEGF + CA group was slower than in other groups. The ANOVA showed that there were significant differences among the groups at weeks 3, 6, and 12 (P G 0.05). The LSD method proved that the mean graft mass in the VEGF + CA group was significantly higher than in other groups at weeks 3, 6, and 12 (P G 0.05). Moreover, the CA group was also significantly higher than the VEGF group at week 12 (P G 0.05) and was significantly higher than the blank control group at weeks 3 and 12 (P G 0.05).
Vascular Density Endothelial cells appeared brownish yellow when observed under the microscope (Eclipse 80i; Nikon) after the sections were immunohistochemically stained with anti-CD34 antibody. Neovessels appeared as single endothelial cell, cell mass, or streaklike vascular. Typical vascular lumens containing erythrocytes were also found (Fig. 11). Table 2 displayed the vascular density measured by image analysis software (valued by relative area). Figure 12 showed that * 2014 Wolters Kluwer Health, Inc. All rights reserved.
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FIGURE 5. Histological observation of grafts obtained at the third week, HE stained. The grafts were surrounded by a thin rim of fibrous tissue. Vascular invasion could be seen in all 4 groups. Focal necrosis in the VEGF + CA group was less common than in other groups. The form and the size of adipocytes in the VEGF + CA group were relatively uniform whereas varied in other groups.
FIGURE 6. Histological observation of grafts obtained at the sixth week, HE stained. The number of vessels increased. Ingrowth of fibrous tissue could be seen in every group, especially in the VEGF group and the blank control group. Necrotic areas were even larger than 3 weeks ago. * 2014 Wolters Kluwer Health, Inc. All rights reserved
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FIGURE 7. Histological observation of grafts obtained at the 12th week, HE stained. Larger necrotic areas (with or without punctate calcification) and less severe fibrosis were observed in the VEGF + CA group than in other groups. There were more fat cells that survived in the VEGF + CA group, whereas only small clusters of fat cells survived in other groups.
the vascular density in the VEGF + CA group increased faster than in the controls. The ANOVA showed that, at the third week, no significant difference was found among the groups (P 9 0.05). At weeks 6 and 12, there are significant differences among the groups (P G 0.05). The LSD method proved that, at the third week, the VEGF + CA group had no significant differences with other groups (P 9 0.05). At the sixth week, MVD in the VEGF + CA group was significantly higher than in the VEGF group and the blank control group (P G 0.05), but no significant differences existed between the VEGF + CA group and the CA group (P 9 0.05). At the 12th week, the MVD in the VEGF + CA group was significantly
higher than in all controls (P 9 0.05); no statistical difference existed within the 3 control groups.
DISCUSSION Many studies concerning adipose tissue transplantation in recent years had quite clearly indicated that revascularization plays an important role in the survival state of adipose graft. In addition, the effect of VEGF or some other angiogenic factors on revascularization of adipose grafts were highly valued.16,18,29,30 Langer et al31 used intravital f luorescent microscopy to observe the functional vessel density of transplanted adipose tissue in hamsters in a quantitative
FIGURE 8. There are CA microspheres surrounding the adipocytes when observed with scanning electron microscope. The edges between cells were indistinct. 648
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FIGURE 9. Transmission electron microscopy images of the grafts at the third and sixth weeks. Carrier materials can be obviously seen outside the adipocytes. No ingested CA microsphere was noticed inside the adipocytes. No distinct fibrous tissue was noticed covering the surface of adipocytes or CA microspheres too.
way: at the border zone of the graft, vascular sprouts were observed already at day 1 after transplantation. In the center of the graft, new vessels appeared on day 3 after transplantation. Functional vessel density increased constantly and reached values that were, at day 12 after transplantation, comparable with those of the fat tissue in situ. Nishimura et al32 suggested that angiogenic factors, including VEGF, revascularized the graft after roughly 7 days. Grafts are nourished via diffusion for the first 4 days. Fat necrosis occurs until revascularization is accomplished. However, apoptosis continues. After the fourth day after grafting, progressive microvascular invasion starts. Topcu et al1 indicated that, at precondition, the recipient bed with VEGF has better survival of fat graft. Placing the graft in a wellvascularized bed is a logical strategy to increase graft take. Vascular endothelial growth factor is a key regulator of physiological angiogenesis. Among all its isoforms, VEGF165 is the most prominent form and has been widely used. This isoform binds to the proteoglycans and heparin in the extracellular matrix medium providing the longest half-life.33 In this study, VEGF165 was loaded by CA microsphere to realize controlled release. By this way, sustainedreleased VEGF promoted the revascularization in the early period after transplantation. As a result, more fat tissue survived in the VEGF + CA group than in other groups. Calcium alginate microspheres were chosen as the controlledrelease carriers because alginate has the advantages of fine biological compatibility, easy preparation, and low cost. In addition, they were also proven to be ideal controlled-release carriers for secreted protein while helping the vascularization itself.27,33 Traditional ways of increasing adipocyte survival are to obtain adipose tissue with minimally invasive surgical techniques and to purify it in a right way. To minimize the trauma of adipocytes, the adipose tissue used in our study was suctioned by a syringe, with a 2.5-mm caliber needle. Although not routinely applied in the clinical setting, in our experiment, fat granules were treated with collagenase, for the
clumped adipocytes to disperse and become evenly distributed. As a result, each injection contained the same amount of adipocytes. The images of sections show that, in the VEGF + CA group, the proliferation of endotheliocytes among the adipose tissue was significantly enhanced and the amount and the caliber of vessels were increased over time. Those factors contributed to a better survival of adipose grafts. The statistical results show that there is no statistically significant difference on MVD between the VEGF + CA group and the CA group until the 12th week, probably because subcutaneous area in mice is a relatively avascular plane and time is needed for vascular ingrowth. In addition, CA microspheres functioned as temporary scaffold in vivo, which also benefited the proliferation of endotheliocytes. As CA disintegrated over time, vessels lost their scaffold. For that reason, the difference between the 2 groups was finally expanded (P G 0.05). The statistical results of graft mass show that the VEGF + CA group was distinctly higher than the controls within the 3 periods. That is because the necrosis of adipose tissue was reduced by the active neovascularization in the VEGF + CA group and because survival of implanted cells strongly relies on sufficient oxygen and nutrient supply. There was no significant difference between the VEGF group and the blank control group within the 3 periods (P 9 0.05). This result supported the point that free VEGF can barely exert its biological function when applied directly. We expected that a higher MVD value would result in more adipocyte survival. However, Figures 10 and 12 implied that the fat grafts were 2 to 3 times heavier after 3 weeks in the VEGF + CA group, whereas there is no significant difference in MVD value at
TABLE 1. Graft Masses at Weeks 3, 6, and 12 (Mean [SD]) VEGF + CA group CA group VEGF group Blank control group
3 wk, mg
6 wk, mg
12 wk, mg
46.0 (16.6) 26.3 (9.4) 22.3 (11.5) 13.0 (9.2)
38.5 (17.3) 23.3 (16.0) 9.5 (8.8) 16.8 (9.7)
29.6 (9.9) 20.5 (11.0) 8.5 (7.0) 6.5 (6.7)
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FIGURE 10. This figure shows the time-varying trend of the grafts’ masses in different groups. The mass decrease in the VEGF + CA group was slower than in other groups. www.annalsplasticsurgery.com
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FIGURE 11. Image of grafts obtained at the sixth week, immunohistochemical stained for endothelial cells. Endothelial cells appeared brownish yellow. Neovessels appeared as single endothelial cell, cell mass, or streaklike vascular. Typical vascular lumens containing erythrocytes were also found.
that point. First, we would like to explain why grafts in the VEGF + CA group are heavier than in other groups, as follows: (1) The survival of adipose graft strongly relies on sufficient oxygen and nutrient supply. In the VEGF + CA group, the controlled release of VEGF caused active neovascularization (although the neovascularization was not distinct at the earlier stage), and higher volume of the graft survived. (2) From the grafts harvested after 3 weeks (Fig. 4), we observed that the CA microspheres in the VEGF + CA group and the CA group were only partly degraded. Many CA microspheres remained, and this also contributed to the great mass of the 2 groups. Second, we would like to explain why the neovascularization (MVD) of the 4 groups are similar: (1) The MVD value increased constantly during the 12 weeks almost in the 4 groups, especially in the VEGF + CA group. The MVD value of the VEGF + CA group would not be very high at the earlier stage (at the third week). (2) Only surviving tissue, in which there must be neovascularization at a rate high enough to provide essential nutrients and oxygen, were harvested to measure MVD value. The poorly nourished adipocytes were dead in the earlier time. Thus, the MVD value of the 3 control groups would
not be very low. (3) Because of the abovementioned reasons, the MVD values were similar between the experimental group and the other groups at the third week. The absorbance of the VEGF + CA group was approximately 80.2% to 90.1% by the 12th week, which was much lower than those reported in the current literature (25%Y80%). That may be partly because there is poor blood supply of subcutaneous area in the rat dorsum and also because human adipocytes cannot adapt to the microenvironment of nude mouse well.34,35 Research about oncological safety of VEGF is needed before human clinical trial is conducted because VEGF is a key regulator of tumor angiogenesis, inducing proliferation, differentiation, and migration of endothelial cells.36
TABLE 2. Relative Area of Vessels per Field Stained With CD34 Immunohistochemical Method (Mean [SD]) VEGF + CA group CA group VEGF group Blank control group
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3 wk
6 wk
12 wk
10,955 (9,523) 11,996 (8,794) 6,364 (4,781) 9,729 (4,260)
29,594 (12,916) 19,785 (13,398) 11,738 (6,674) 13,196 (6,925)
44,149 (25,135) 24,507 (8,568) 12,318 (8,906) 8,026 (4,638)
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FIGURE 12. The time-varying trend of vascular density of grafts is shown in this figure. The vascular density in the VEGF + CA group increased faster than in the controls. * 2014 Wolters Kluwer Health, Inc. All rights reserved.
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CONCLUSIONS Calcium alginate microspheres loaded with VEGF can significantly promote the neovascularization of adipose graft and improve the survival rate of adipocytes. REFERENCES 1. Topcu A, Aydin OE, Unlu M, et al. Increasing the viability of fat grafts by vascular endothelial growth factor. Arch Facial Plast Surg. 2012;14:270Y276. 2. Coleman SR. Structural fat grafts: the ideal filler? Clin Plast Surg. 2001;28: 111Y119. 3. Leuchter I, Schweizer V, Hohlfeld J, et al. Treatment of velopharyngeal insufficiency by autologous fat injection. Eur Arch Otorhinolaryngol. 2010;267: 977Y983. 4. Trojahn Kolle SF, Oliveri RS, Glovinski PV, et al. Importance of mesenchymal stem cells in autologous fat grafting: a systematic review of existing studies. J Plast Surg Hand Surg. 2012;46:59Y68. 5. Sinna R, Delay E, Garson S, et al. Breast fat grafting (lipomodelling) after extended latissimus dorsi flap breast reconstruction: a preliminary report of 200 consecutive cases. J Plast Reconstr Aesthet Surg. 2010;63:1769Y1777. 6. Delay E, Garson S, Tousson G, et al. Fat injection to the breast: technique, results, and indications based on 880 procedures over 10 years. Aesthet Surg J. 2009;29:360Y376. 7. Missana MC, Laurent I, Barreau L, et al. Autologous fat transfer in reconstructive breast surgery: indications, technique and results. Eur J Surg Oncol. 2007;33:685Y690. 8. Kononas TC, Bucky LP, Hurley C, et al. The fate of suctioned and surgically removed fat after reimplantation for soft-tissue augmentation: a volumetric and histologic study in the rabbit. Plast Reconstr Surg. 1993;91:763Y768. 9. Carraway JH, Mellow CG. Syringe aspiration and fat concentration: a simple technique for autologous fat injection. Ann Plast Surg. 1990;24:293Y296; discussion 297. 10. Mikus JL, Koufman JA, Kilpatrick SE. Fate of liposuctioned and purified autologous fat injections in the canine vocal fold. Laryngoscope. 1995; 105:17Y22. 11. Ellenbogen R. Free autogenous pearl fat grafts in the faceVa preliminary report of a rediscovered technique. Ann Plast Surg. 1986;16:179Y194. 12. Bircoll M, Novack BH. Autologous fat transplantation employing liposuction techniques. Ann Plast Surg. 1987;18:327Y329. 13. Har-Shai Y, Lindenbaum ES, Gamliel-Lazarovich A, et al. An integrated approach for increasing the survival of autologous fat grafts in the treatment of contour defects. Plast Reconstr Surg. 1999;104:945Y954. 14. Yuksel E, Weinfeld AB, Cleek R, et al. Increased free fat-graft survival with the long-term, local delivery of insulin, insulin-like growth factor-I, and basic fibroblast growth factor by PLGA/PEG microspheres. Plast Reconstr Surg. 2000;105:1712Y1720. 15. Borges J, Torio-Padron N, Momeni A, et al. Adipose precursor cells (preadipocytes) induce formation of new vessels in fibrin glue on the newly developed cylinder chorioallantoic membrane model (CAM). Minim Invasive Ther Allied Technol. 2006;15:246Y252. 16. Yi CG, Xia W, Zhang LX, et al. VEGF gene therapy for the survival of transplanted fat tissue in nude mice. J Plast Reconstr Aesthet Surg. 2007;60: 272Y278.
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17. Bartynski J, Marion MS, Wang TD. Histopathologic evaluation of adipose autografts in a rabbit ear model. Otolaryngol Head Neck Surg. 1990;102: 314Y321. 18. Fukumura D, Ushiyama A, Duda DG, et al. Paracrine regulation of angiogenesis and adipocyte differentiation during in vivo adipogenesis. Circ Res. 2003;93:e88Ye97. 19. Zhao J, Yi C, Li L, et al. Observations on the survival and neovascularization of fat grafts interchanged between C57BL/6-gfp and C57BL/6 mice. Plast Reconstr Surg. 2012;130:398eY406e. 20. Nillesen ST, Geutjes PJ, Wismans R, et al. Increased angiogenesis and blood vessel maturation in acellular collagen-heparin scaffolds containing both FGF2 and VEGF. Biomaterials. 2007;28:1123Y1131. 21. Carmeliet P. Angiogenesis in health and disease. Nat Med. 2003;9:653Y660. 22. Gianni-Barrera R, Trani M, Reginato S, et al. To sprout or to split? VEGF, notch and vascular morphogenesis. Biochem Soc Trans. 2011;39:1644Y1648. 23. Kim DC, Sugiyama Y, Satoh H, et al. Kinetic analysis of in vivo receptordependent binding of human epidermal growth factor by rat tissues. J Pharm Sci. 1988;77:200Y207. 24. Konturek SJ, Pawlik W, Mysh W, et al. Comparison of organ uptake and disappearance half-time of human epidermal growth factor and insulin. Regul Pept. 1990;30:137Y148. 25. Losordo DW, Dimmeler S. Therapeutic angiogenesis and vasculogenesis for ischemic disease part I: angiogenic cytokines. Circulation. 2004;109: 2487Y2491. 26. Braccini I, Perez S. Molecular basis of C(2+)-induced gelation in alginates and pectins: the egg-box model revisited. Biomacromolecules. 2001;2:1089Y1096. 27. Rokstad AM, Holtan S, Strand B, et al. Microencapsulation of cells producing therapeutic proteins: optimizing cell growth and secretion. Cell Transplant. 2002;11:313Y324. 28. Yao R, Zhang R, Luan J, et al. Alginate and alginate/gelatin microspheres for human adipose-derived stem cell encapsulation and differentiation. Biofabrication. 2012;4:025007. 29. Aoki S, Toda S, Sakemi T, et al. Coculture of endothelial cells and mature adipocytes actively promotes immature preadipocyte development in vitro. Cell Struct Funct. 2003;28:55Y60. 30. Hutley LJ, Herington AC, Shurety W, et al. Human adipose tissue endothelial cells promote preadipocyte proliferation. Am J Physiol Endocrinol Metab. 2001;281:E1037YE1044. 31. Langer S, Sinitsina I, Biberthaler P, et al. Revascularization of transplanted adipose tissue: a study in the dorsal skinfold chamber of hamsters. Ann Plast Surg. 2002;48:53Y59. 32. Nishimura T, Hashimoto H, Nakanishi I, et al. Microvascular angiogenesis and apoptosis in the survival of free fat grafts. Laryngoscope. 2000;110: 1333Y1338. 33. Ferrara N, Gerber HP, LeCouter J. The biology of VEGF and its receptors. Nat Med. 2003;9:669Y676. 34. Eppley BL, Sadove AM. A physicochemical approach to improving free fat graft survival: preliminary observations. Aesthetic Plast Surg. 1991;15: 215Y218. 35. Eppley BL, Sidner RA, Platis JM, et al. Bioactivation of free-fat transfers: a potential new approach to improving graft survival. Plast Reconstr Surg. 1992;90:1022Y1030. 36. Van der Veldt AA, Lubberink M, Bahce I, et al. Rapid decrease in delivery of chemotherapy to tumors after anti-VEGF therapy: implications for scheduling of anti-angiogenic drugs. Cancer Cell. 2012;21:82Y91.
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Effect of Calcium Alginate Microsphere Loaded With Vascular Endothelial Growth Factor on Adipose Tissue Transplantation Shi-Li Ding, MD, Meng-Yuan Zhang, MD, Song-Jia Tang, MD, Hu Yang, MD, and Wei-Qiang Tan, MD Abstract: Revascularization in the early period after transplantation is the key to improving adipocyte survival. Vascular endothelial growth factor (VEGF) is known as the master regulator of angiogenesis. However, consensus is lacking regarding safe and efficient methods for applying VEGF in free fat transplantation in the clinical setting. We constructed calcium alginate (CA) microspheres loaded with VEGF to increase the survival of implanted adipocytes. BALB/c nude mice were used as adipose tissue transplantation receptors. Adipocytes were mixed with CA microspheres loaded with VEGF and implanted subcutaneously into the dorsum of mice. Grafts were harvested at week 3, 6, and 12 after transplantation. We found that the mass and microvascular density of grafts in the VEGF + CA group (CA microspheres loaded with VEGF) were statistically higher than that of other groups in a timedependent manner. We demonstrated that CA microspheres loaded with VEGF can significantly promote the fat graft neovascularization, thus improving adipocyte survival. Key Words: revascularization, vascular endothelial growth factor, calcium alginate, free fat transplantation (Ann Plast Surg 2015;75: 644Y651)
A
utologous adipose tissue is usually considered an ideal filler material in plastic surgery because it is versatile, natural-appearing, inexpensive, biocompatible, and available in a sufficient amount.1Y4 Furthermore, the application of injection method gives autologous adipose tissue transplantation the advantages of simple manipulation and minimal trauma. Clinically, this technique has been widely used in correcting surface depression or augmenting soft tissue. However, with an absorbance of 25% to 80%,4Y8 the unpredictable and often low survival of the autologous fat tissue affects the outcome of this technique. In the past 20 years, many studies have been done to increase the viability of autogenous fat tissue.9Y18 The view that a fast blood supply reestablishment is the key to improving adipocyte survival has been widely accepted. Thereby, vascular endothelial growth factor (VEGF) became the research hot topic.13,19,20 Vascular endothelial growth factor is the master regulator of angiogenesis both in physical and pathological development.21 It is Received August 29, 2013, and accepted for publication, after revision, February 12, 2014. From the Department of Plastic Surgery, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, People’s Republic of China. Dr Ding and Dr Zhang contributed equally to this article as first authors. Conflicts of interest and sources of funding: This work was supported by funding from the National Natural Science Foundation of China (No. 81372072 and 30800228), Zhejiang Provincial Medical and Healthy Science Foundation of China (No. 2013RCB004), and Zhejiang Provincial Natural Science Foundation of China (No. LY12H15006). Reprints: Wei Qiang Tan, MD, Department of Plastic Surgery, The First Affiliated Hospital, College of Medicine, Zhejiang University, 79 Qingchun Rd, Hangzhou, Zhejiang Province 310003, People’s Republic of China. E-mail: [email protected]. Copyright * 2014 Wolters Kluwer Health, Inc. All rights reserved. ISSN: 0148-7043/15/7506-0644 DOI: 10.1097/SAP.0000000000000201
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capable of starting the complex cascade of events leading to endothelial cell activation, assembly of new vascular structures, mural cell recruitment, and vessel stabilization.22 However, the incorporation of growth factors involves many difficulties because of their short half-lives of only several minutes in circulation.23,24 Free VEGF can barely exert its biological function when applied directly25 because it tends to diffuse, dilute, and degrade, resulting in very low bioavailability. We applied calcium alginate (CA) as drug carrier to achieve a controlled release of VEGF. Alginates are a family of natural anionic and biocompatible polysaccharides derived from brown algae. They are very suitable for encapsulating biomacromolecules and living cells because of their low toxicity, high biocompatibility, and timely degradation.26Y28 We constructed CA microspheres loaded with VEGF to improve the bioavailability of VEGF. In addition, we mixed them with implanted adipocytes to increase adipocyte survival rate. In our animal experiment, the mixture was injected subcutaneously into the dorsa of nude mice. The in vivo angiogenic response to the controlledrelease system was measured by evaluating the microvessel density (MVD) with immunohistochemistry.
MATERIALS AND METHODS The animal experiment designed in this study was approved by the Ethics Committee of Zhejiang University.
Preparation of CA Microspheres Loaded With VEGF A solution of 1.2% sodium alginate (wt/vol; Sigma, San Diego, Calif ) in 0.9% (wt/vol) sodium chloride was magnetic stirred for 2 hours until completely dissolved. Then, the solution was sterilized by a high-pressure sterilizer for 40 minutes at 121-C. After that, it was dried and preserved at 4-C for later procedure. One milliliter of VEGF165 (1 Kg/mL; Peprotech, Rocky Hill, NJ) was added into 4 mL of sodium alginate solution to obtain 5 mL of VEGF solution (0.2 Kg/mL). Then, the dilution was slowly extruded dropwise through a 23-gauge hypodermic needle into 10 mL of calcium chloride solution (11.3 g/L). Wait for 10 minutes at room temperature to reach a good cross-linking effect. Then, the rest of the calcium chloride solution was removed, and the microspheres were rinsed for 5 times with 0.9% (wt/vol) sodium chloride (Fig. 1).
Adipose Tissue Transplantation A liposuction needle (2.5-mm caliber) was connected to a 20-mL syringe. Adipose tissue was suctioned subcutaneously from the abdomen of a healthy young female patient. Collagenase I (Sigma, St Louis, Mo) was dissolved with Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Carlsbad, NM) in a 37-C thermostatic water bath and filled into 16 centrifuge tubes, 4 mL each (1.0 g/L). Two milliliters of adipose tissue (32 mL in total) was filled into each centrifuge tube. The adipose tissue was digested for 1 hour. After that, each tube was filled Annals of Plastic Surgery
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FIGURE 3. The grafts were distributed well proportioned to avoid merging.
FIGURE 1. The CA microspheres can be seen attaching to the surface of the centrifuge tube.
with 6 mL of DMEM (supplemented with 10% fetal bovine serum) to terminate the digestion. Then, the mixtures were centrifuged at 1200 rpm for 10 minutes. The mixtures appeared as 4 layers from top to bottom: lipid, adipocytes, supernatant, and pellet (Fig. 2). Adipocytes were carefully preserved for subsequent procedure. A total of 4.5 mL of adipocytes was mixed with 1.5 mL of the following materials before transplantation: CA microspheres loaded with VEGF (0.2 Kg/mL), CA microspheres, VEGF (0.2 Kg/mL), and DMEM. Six- to 8-week-old BALB/c nude mice weighing 20 to 25 g were selected regardless of sex (provided by the experimental animal center of Zhejiang University). Twenty-four mice were equally divided into 4 cages at random. The abovementioned adipose grafts were implanted subcutaneously into the dorsum of each mouse at 4 points with a sharp 12gauge needle attached to a 1-mL syringe. Each point was injected with 0.2 mL of the graft; the adipocyte amount was 3 105 per site. The grafts of different composition were assigned to the following groups: VEGF + CA group (adipocytes and CA microspheres loaded with VEGF), CA group (adipocytes and blank CA microspheres), VEGF group (adipocytes and free VEGF), and blank control group
(adipocytes only). The grafts were distributed well proportioned to avoid merging (Fig. 3). By this way, the 4 grafts in each mouse were set symmetrically, and their sites were rotated clockwise to eliminate potential skewing of results. Mice in the same cage shared the same order of injection sites.
Sample Collection and Histopathological Measurement Two mice in each cage were killed at weeks 3, 6, and 12 after transplantation. The fat grafts were carefully stripped out and weighed. The exterior characteristics of the graft were visually observed. Sections of grafts were observed under a light microscope (Eclipse 80i; Nikon) after staining with hematoxylin and eosin (HE). Observation of adipocytes, degree of inf lammation, and fibrosis were recorded. The grafts harvested at the third and sixth weeks of the VEGF + CA group were sent for scanning electron microscopy and transmission electron microscopy.
Microvascular Density Determination Samples were paraffin embedded and sliced into 4-Km sections. The expression of CD34 was detected by means of immunohistochemical staining. Microvessels among the adipocytes were observed under a light microscope (Eclipse 80i; Nikon) at low magnification (100) to find the area where the highest vascular density was located. Then, 4 fields of fixed magnification (400) were chosen randomly, and images were saved. The relative area of stained microvessel in each image was calculated by Image-Pro Plus 6.0 software (Media Cybernetics; Rockville, Md).
Statistical Analysis Data are presented as mean (SD). All data were analyzed with the Statistical Package for the Social Sciences 13.0 software (IBM, Chicago, Ill). Comparisons of means of several groups were tested with 1-way analysis of variance (ANOVA). Comparisons of survival between every 2 groups at different times were tested by least significant difference (LSD) method. Probability values of P G 0.05 were interpreted to denote statistical significance.
RESULTS All animals tolerated the operations well. There was no evidence of any ulceration, infection, hematoma, or seroma at the injection points.
General Observation of Adipose Grafts FIGURE 2. The mixture appeared as 4 layers from top to bottom: lipid, adipocytes, supernatant, and pellet. * 2014 Wolters Kluwer Health, Inc. All rights reserved
The grafts were yellow, soft, and easily dissectible. They also had well-defined periphery with intact envelope (Fig. 4). Vessels were visible from the grafts’ surfaces. There were more vessels on the www.annalsplasticsurgery.com
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FIGURE 4. A graft harvested from the dorsum of a mouse at the third week in the VEGF + CA group (upper left). The graft is made up of yellow adipose tissue and transparent CA microspheres. The other 3 pictures are the grafts harvested at weeks 3, 6, and 12. The grafts were yellow, soft, and easily dissectible. They also had well-defined periphery with intact envelope. Vessels were visible from the surface of grafts. There were more vessels on the surface of grafts of the VEGF + CA group than other groups. Not completely absorbed CA can be seen from the grafts that were harvested at the third week. Grafts in the VEGF + CA group have a larger volume than grafts in other groups in all periods.
surface of the grafts of the VEGF + CA group than other groups. Fat liquefaction happened among the grafts individually. Not completely absorbed CA can be seen from the grafts that were harvested at the third week.
Histological Evaluation At the third week, the grafts were surrounded by a thin envelope of fibrous tissue. Inf lammatory cellular infiltration was not found in or around the adipose tissue. Vascular invasion could be seen in all 4 groups. Focal necrosis in the VEGF + CA group was less common than in other groups. The form and the size of adipocytes in the VEGF + CA group were relatively uniform whereas varied in other groups (Fig. 5). At the sixth week, the number of vessels increased. Ingrowth of fibrous tissue could be seen in every group, especially in the VEGF group and the blank control group. Necrotic areas were even larger than 3 weeks ago (Fig. 6). In the grafts obtained at the 12th week, larger necrotic areas (with or without punctate calcification) and less severe fibrosis were observed in the VEGF + CA group than in other groups. There were more fat cells that survived in the VEGF + CA group, whereas only small clusters of fat cells survived in other groups (Fig. 7).
Electron Microscopy Observation There are CA microspheres surrounding the adipocytes in the sections of both the third week and the sixth week when observed with scanning electron microscope. The edges between cells were indistinct (Fig. 8). Calcium alginate microspheres can be obviously seen 646
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outside the adipocytes, viewed with transmission electron microscope. No incepted CA was noticed inside the adipocytes. No distinct fibrous tissue was noticed covering the surfaces of adipocytes or CA microspheres too (Fig. 9).
Graft Mass The graft masses of the 4 groups at weeks 3, 6, and 12 were shown in Table 1. Figure 10 showed that the mass decrease in the VEGF + CA group was slower than in other groups. The ANOVA showed that there were significant differences among the groups at weeks 3, 6, and 12 (P G 0.05). The LSD method proved that the mean graft mass in the VEGF + CA group was significantly higher than in other groups at weeks 3, 6, and 12 (P G 0.05). Moreover, the CA group was also significantly higher than the VEGF group at week 12 (P G 0.05) and was significantly higher than the blank control group at weeks 3 and 12 (P G 0.05).
Vascular Density Endothelial cells appeared brownish yellow when observed under the microscope (Eclipse 80i; Nikon) after the sections were immunohistochemically stained with anti-CD34 antibody. Neovessels appeared as single endothelial cell, cell mass, or streaklike vascular. Typical vascular lumens containing erythrocytes were also found (Fig. 11). Table 2 displayed the vascular density measured by image analysis software (valued by relative area). Figure 12 showed that * 2014 Wolters Kluwer Health, Inc. All rights reserved.
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FIGURE 5. Histological observation of grafts obtained at the third week, HE stained. The grafts were surrounded by a thin rim of fibrous tissue. Vascular invasion could be seen in all 4 groups. Focal necrosis in the VEGF + CA group was less common than in other groups. The form and the size of adipocytes in the VEGF + CA group were relatively uniform whereas varied in other groups.
FIGURE 6. Histological observation of grafts obtained at the sixth week, HE stained. The number of vessels increased. Ingrowth of fibrous tissue could be seen in every group, especially in the VEGF group and the blank control group. Necrotic areas were even larger than 3 weeks ago. * 2014 Wolters Kluwer Health, Inc. All rights reserved
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FIGURE 7. Histological observation of grafts obtained at the 12th week, HE stained. Larger necrotic areas (with or without punctate calcification) and less severe fibrosis were observed in the VEGF + CA group than in other groups. There were more fat cells that survived in the VEGF + CA group, whereas only small clusters of fat cells survived in other groups.
the vascular density in the VEGF + CA group increased faster than in the controls. The ANOVA showed that, at the third week, no significant difference was found among the groups (P 9 0.05). At weeks 6 and 12, there are significant differences among the groups (P G 0.05). The LSD method proved that, at the third week, the VEGF + CA group had no significant differences with other groups (P 9 0.05). At the sixth week, MVD in the VEGF + CA group was significantly higher than in the VEGF group and the blank control group (P G 0.05), but no significant differences existed between the VEGF + CA group and the CA group (P 9 0.05). At the 12th week, the MVD in the VEGF + CA group was significantly
higher than in all controls (P 9 0.05); no statistical difference existed within the 3 control groups.
DISCUSSION Many studies concerning adipose tissue transplantation in recent years had quite clearly indicated that revascularization plays an important role in the survival state of adipose graft. In addition, the effect of VEGF or some other angiogenic factors on revascularization of adipose grafts were highly valued.16,18,29,30 Langer et al31 used intravital f luorescent microscopy to observe the functional vessel density of transplanted adipose tissue in hamsters in a quantitative
FIGURE 8. There are CA microspheres surrounding the adipocytes when observed with scanning electron microscope. The edges between cells were indistinct. 648
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FIGURE 9. Transmission electron microscopy images of the grafts at the third and sixth weeks. Carrier materials can be obviously seen outside the adipocytes. No ingested CA microsphere was noticed inside the adipocytes. No distinct fibrous tissue was noticed covering the surface of adipocytes or CA microspheres too.
way: at the border zone of the graft, vascular sprouts were observed already at day 1 after transplantation. In the center of the graft, new vessels appeared on day 3 after transplantation. Functional vessel density increased constantly and reached values that were, at day 12 after transplantation, comparable with those of the fat tissue in situ. Nishimura et al32 suggested that angiogenic factors, including VEGF, revascularized the graft after roughly 7 days. Grafts are nourished via diffusion for the first 4 days. Fat necrosis occurs until revascularization is accomplished. However, apoptosis continues. After the fourth day after grafting, progressive microvascular invasion starts. Topcu et al1 indicated that, at precondition, the recipient bed with VEGF has better survival of fat graft. Placing the graft in a wellvascularized bed is a logical strategy to increase graft take. Vascular endothelial growth factor is a key regulator of physiological angiogenesis. Among all its isoforms, VEGF165 is the most prominent form and has been widely used. This isoform binds to the proteoglycans and heparin in the extracellular matrix medium providing the longest half-life.33 In this study, VEGF165 was loaded by CA microsphere to realize controlled release. By this way, sustainedreleased VEGF promoted the revascularization in the early period after transplantation. As a result, more fat tissue survived in the VEGF + CA group than in other groups. Calcium alginate microspheres were chosen as the controlledrelease carriers because alginate has the advantages of fine biological compatibility, easy preparation, and low cost. In addition, they were also proven to be ideal controlled-release carriers for secreted protein while helping the vascularization itself.27,33 Traditional ways of increasing adipocyte survival are to obtain adipose tissue with minimally invasive surgical techniques and to purify it in a right way. To minimize the trauma of adipocytes, the adipose tissue used in our study was suctioned by a syringe, with a 2.5-mm caliber needle. Although not routinely applied in the clinical setting, in our experiment, fat granules were treated with collagenase, for the
clumped adipocytes to disperse and become evenly distributed. As a result, each injection contained the same amount of adipocytes. The images of sections show that, in the VEGF + CA group, the proliferation of endotheliocytes among the adipose tissue was significantly enhanced and the amount and the caliber of vessels were increased over time. Those factors contributed to a better survival of adipose grafts. The statistical results show that there is no statistically significant difference on MVD between the VEGF + CA group and the CA group until the 12th week, probably because subcutaneous area in mice is a relatively avascular plane and time is needed for vascular ingrowth. In addition, CA microspheres functioned as temporary scaffold in vivo, which also benefited the proliferation of endotheliocytes. As CA disintegrated over time, vessels lost their scaffold. For that reason, the difference between the 2 groups was finally expanded (P G 0.05). The statistical results of graft mass show that the VEGF + CA group was distinctly higher than the controls within the 3 periods. That is because the necrosis of adipose tissue was reduced by the active neovascularization in the VEGF + CA group and because survival of implanted cells strongly relies on sufficient oxygen and nutrient supply. There was no significant difference between the VEGF group and the blank control group within the 3 periods (P 9 0.05). This result supported the point that free VEGF can barely exert its biological function when applied directly. We expected that a higher MVD value would result in more adipocyte survival. However, Figures 10 and 12 implied that the fat grafts were 2 to 3 times heavier after 3 weeks in the VEGF + CA group, whereas there is no significant difference in MVD value at
TABLE 1. Graft Masses at Weeks 3, 6, and 12 (Mean [SD]) VEGF + CA group CA group VEGF group Blank control group
3 wk, mg
6 wk, mg
12 wk, mg
46.0 (16.6) 26.3 (9.4) 22.3 (11.5) 13.0 (9.2)
38.5 (17.3) 23.3 (16.0) 9.5 (8.8) 16.8 (9.7)
29.6 (9.9) 20.5 (11.0) 8.5 (7.0) 6.5 (6.7)
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FIGURE 10. This figure shows the time-varying trend of the grafts’ masses in different groups. The mass decrease in the VEGF + CA group was slower than in other groups. www.annalsplasticsurgery.com
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FIGURE 11. Image of grafts obtained at the sixth week, immunohistochemical stained for endothelial cells. Endothelial cells appeared brownish yellow. Neovessels appeared as single endothelial cell, cell mass, or streaklike vascular. Typical vascular lumens containing erythrocytes were also found.
that point. First, we would like to explain why grafts in the VEGF + CA group are heavier than in other groups, as follows: (1) The survival of adipose graft strongly relies on sufficient oxygen and nutrient supply. In the VEGF + CA group, the controlled release of VEGF caused active neovascularization (although the neovascularization was not distinct at the earlier stage), and higher volume of the graft survived. (2) From the grafts harvested after 3 weeks (Fig. 4), we observed that the CA microspheres in the VEGF + CA group and the CA group were only partly degraded. Many CA microspheres remained, and this also contributed to the great mass of the 2 groups. Second, we would like to explain why the neovascularization (MVD) of the 4 groups are similar: (1) The MVD value increased constantly during the 12 weeks almost in the 4 groups, especially in the VEGF + CA group. The MVD value of the VEGF + CA group would not be very high at the earlier stage (at the third week). (2) Only surviving tissue, in which there must be neovascularization at a rate high enough to provide essential nutrients and oxygen, were harvested to measure MVD value. The poorly nourished adipocytes were dead in the earlier time. Thus, the MVD value of the 3 control groups would
not be very low. (3) Because of the abovementioned reasons, the MVD values were similar between the experimental group and the other groups at the third week. The absorbance of the VEGF + CA group was approximately 80.2% to 90.1% by the 12th week, which was much lower than those reported in the current literature (25%Y80%). That may be partly because there is poor blood supply of subcutaneous area in the rat dorsum and also because human adipocytes cannot adapt to the microenvironment of nude mouse well.34,35 Research about oncological safety of VEGF is needed before human clinical trial is conducted because VEGF is a key regulator of tumor angiogenesis, inducing proliferation, differentiation, and migration of endothelial cells.36
TABLE 2. Relative Area of Vessels per Field Stained With CD34 Immunohistochemical Method (Mean [SD]) VEGF + CA group CA group VEGF group Blank control group
650
3 wk
6 wk
12 wk
10,955 (9,523) 11,996 (8,794) 6,364 (4,781) 9,729 (4,260)
29,594 (12,916) 19,785 (13,398) 11,738 (6,674) 13,196 (6,925)
44,149 (25,135) 24,507 (8,568) 12,318 (8,906) 8,026 (4,638)
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FIGURE 12. The time-varying trend of vascular density of grafts is shown in this figure. The vascular density in the VEGF + CA group increased faster than in the controls. * 2014 Wolters Kluwer Health, Inc. All rights reserved.
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CONCLUSIONS Calcium alginate microspheres loaded with VEGF can significantly promote the neovascularization of adipose graft and improve the survival rate of adipocytes. REFERENCES 1. Topcu A, Aydin OE, Unlu M, et al. Increasing the viability of fat grafts by vascular endothelial growth factor. Arch Facial Plast Surg. 2012;14:270Y276. 2. Coleman SR. Structural fat grafts: the ideal filler? Clin Plast Surg. 2001;28: 111Y119. 3. Leuchter I, Schweizer V, Hohlfeld J, et al. Treatment of velopharyngeal insufficiency by autologous fat injection. Eur Arch Otorhinolaryngol. 2010;267: 977Y983. 4. Trojahn Kolle SF, Oliveri RS, Glovinski PV, et al. Importance of mesenchymal stem cells in autologous fat grafting: a systematic review of existing studies. J Plast Surg Hand Surg. 2012;46:59Y68. 5. Sinna R, Delay E, Garson S, et al. Breast fat grafting (lipomodelling) after extended latissimus dorsi flap breast reconstruction: a preliminary report of 200 consecutive cases. J Plast Reconstr Aesthet Surg. 2010;63:1769Y1777. 6. Delay E, Garson S, Tousson G, et al. Fat injection to the breast: technique, results, and indications based on 880 procedures over 10 years. Aesthet Surg J. 2009;29:360Y376. 7. Missana MC, Laurent I, Barreau L, et al. Autologous fat transfer in reconstructive breast surgery: indications, technique and results. Eur J Surg Oncol. 2007;33:685Y690. 8. Kononas TC, Bucky LP, Hurley C, et al. The fate of suctioned and surgically removed fat after reimplantation for soft-tissue augmentation: a volumetric and histologic study in the rabbit. Plast Reconstr Surg. 1993;91:763Y768. 9. Carraway JH, Mellow CG. Syringe aspiration and fat concentration: a simple technique for autologous fat injection. Ann Plast Surg. 1990;24:293Y296; discussion 297. 10. Mikus JL, Koufman JA, Kilpatrick SE. Fate of liposuctioned and purified autologous fat injections in the canine vocal fold. Laryngoscope. 1995; 105:17Y22. 11. Ellenbogen R. Free autogenous pearl fat grafts in the faceVa preliminary report of a rediscovered technique. Ann Plast Surg. 1986;16:179Y194. 12. Bircoll M, Novack BH. Autologous fat transplantation employing liposuction techniques. Ann Plast Surg. 1987;18:327Y329. 13. Har-Shai Y, Lindenbaum ES, Gamliel-Lazarovich A, et al. An integrated approach for increasing the survival of autologous fat grafts in the treatment of contour defects. Plast Reconstr Surg. 1999;104:945Y954. 14. Yuksel E, Weinfeld AB, Cleek R, et al. Increased free fat-graft survival with the long-term, local delivery of insulin, insulin-like growth factor-I, and basic fibroblast growth factor by PLGA/PEG microspheres. Plast Reconstr Surg. 2000;105:1712Y1720. 15. Borges J, Torio-Padron N, Momeni A, et al. Adipose precursor cells (preadipocytes) induce formation of new vessels in fibrin glue on the newly developed cylinder chorioallantoic membrane model (CAM). Minim Invasive Ther Allied Technol. 2006;15:246Y252. 16. Yi CG, Xia W, Zhang LX, et al. VEGF gene therapy for the survival of transplanted fat tissue in nude mice. J Plast Reconstr Aesthet Surg. 2007;60: 272Y278.
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