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The Use of Angiogenic Cytokines to Reduce Fat Necrosis in Experimental Tram Flaps
Abdel-naser M. Khallaf, MD, PhD*
Paul D. Smith, MD†
Wyatt G. Payne, MD†
Theodore J. Strickland, M.D†
Francis Ko, BS†
Martin C. Robson, MD†
†The Institute of Tissue Regeneration, Repair, and Rehabilitation, Department of Veterans Affairs Medical Center, Bay Pines, Florida, and Department of Surgery, Division of Plastic Surgery, University of South Florida, Tampa, Florida
†Department of Plastic Surgery, Al-Azhar University, College of Medicine, Naser City, Cairo, Egypt
The work was supported by a grant to the Bay Pines Foundation from the Egyptian Government.
KEY WORDS: Angiogenic Cytokines, TRAM flaps, fat necrosis, fa/fa/ Zucker rats, animal model
Problem: Fat necrosis remains a serious complication in reconstructive flaps. The transverse rectus abdominis (TRAM) flap, which has the desirable amount of adipose tissue for filling and contouring defects, is particularly susceptible to fat necrosis. The incidence and severity of fat necrosis has been decreased by surgical vascular augmentation of TRAM flaps such as delay procedures, use of multiple pedicles, and the use of microvascular anastamoses. This work describes the use of angiogenic cytokines to incite angiogenesis to augment the flap vasculature without additional surgical manipulations.
Method: A newly described experimental model of fat necrosis using TRAM flaps created in genetically obese fa/fa Zucker rats was used to evaluate the effect of angiogenic cytokines on fat necrosis. Eight groups of animals (one with control flaps and seven with various angiogenic cytokines) were used to evaluate clinical palpable firmness of the flaps and the degree of histological inflammation and fat necrosis. Numbers of blood vessels were also evaluated for each group. These evaluations were compared to control flaps and normal unmanipulated abdominal adipose tissue.
Results: All control flaps showed palpable firmness. The cytokine-treated flap groups all had some palpably soft flaps, with four of the groups (GM-CSF, GM-CSF+AII, VEGF165, PD-ECGF) having totally soft flaps in over 85% of the animals. Histologically, four of the cytokine-treated groups showed significantly less inflammation than the control groups (P < .05). All of the cytokine-treated groups showed less histological fat necrosis than the control group (P < .05). All of the experimental groups showed significantly more blood vessels than unmanipulated normal abdominal adipose tissue.
Conclusions: Medical stimulation of angiogenesis as a way to decrease fat necrosis is possible in experimental TRAM flaps. Angiogenic cytokines that directly stimulate angiogenesis without stimulating inflammation appear to have the greatest potential. From the data presented, VEGF165, VGEF121, and PD-ECGF would be the best candidates for further investigation.
A degree of fat necrosis is the usual sequelae when adipose tissue is traumatized or manipulated by a surgical procedure. It can even occur spontaneously in unmanipulated body fat.1 When fat necrosis becomes clinically obvious with a palpable mass, inflammation, drainage, or overlying skin compromise, it becomes a complication. When occurring in a reconstructive flap, fat necrosis can range from a relatively minor spontaneously resolving complication to a major problem resulting in infection and liquefaction that requires surgical drainage.2
Transfer of adequate amounts of adipose tissue is often desirable in plastic surgery such as for breast reconstruction. With the introduction of the transverse rectus abdominis musculocutaneous (TRAM) flap by Hartrampf et al.3 in 1982, large amounts of adipose tissue could be transferred with good predictability. Despite its usual success, the TRAM flap can develop fat necrosis in 12% to 35% of cases.2,4-6 This figure has been reported as high as 40% when specialized mammographic techniques of the flap are used.7
Fat necrosis has been defined as the formation of a small firm area (or areas) of scar tissue caused by ischemic necrosis of subcutaneous fat in the absence of necrosis of the overlying skin.2 Although the precise etiology of fat necrosis remains unknown, it is believed that fat cells subjected to a sublethal ischemic insult may be the initiating step in clinical fat necrosis.6,8 Ischemia in TRAM flaps can be caused by circulatory alterations such as inadequate arterial flow, reduction in venous outflow, or inherent tissue perfusion problems associated with a patient’s condition such as obesity, significant smoking history, diabetes mellitus, or collagen vascular disease.9
Attempts to maximize flap perfusion and minimize ischemia in TRAM flaps have included use of multiple vascular pedicles,10-12 various techniques of delay procedures,13-15 supercharging or turbocharging the flaps,16-18 eliminating possible kinking of pedicles by performing free flaps,19-23 and careful selection of patients with minimal risk factors.9,10,24 Despite decreasing the incidence and severity of fat necrosis in many reported series by these techniques, the complication has not been eliminated.
Attempts to study fat necrosis or to determine ways to prevent or treat this problem have been hindered by lack of an adequate animal model that mimics the human condition. Although a rat rectus abdominis flap has been reported and used to study partial skin loss of the flap, it has not been a model to study fat necrosis because of the sparse subcutaneous fat in the rat.25,26 Our group has recently used the genetically obese leptin receptor-negative fa/fa Zucker rat and developed a reliable model of fat necrosis in a TRAM flap.27 This model consistently produces clinical and histologic changes seen in the clinical scenario. It is the purpose of the present studies to attempt to prevent or minimize the occurrence of fat necrosis in experimental TRAM flaps without surgical augmentation of the vascular supply.
Angiogenesis (the formation of new blood vessels from preexisting blood vessels) is a physiologic process for neovascularization.28-30 It has been reported to be one of the underlying mechanisms of the delay procedure.31,32 Various cytokine growth factors have been shown to stimulate angiogenesis.33 Knighton et al.34 suggested that their mechanisms of action could be either direct by acting on the endothelial cell, or indirect by stimulating inflammation, which in turn elicits angiogenesis. With the advent of recombinant technology, these cytokine growth factors are available for study and potential clinical use. This study will evaluate the effect of angiogenic factors in the fa/fa Zucker rat TRAM model to minimize fat necrosis.
MATERIALS AND METHODS
The animal model used for these experiments was a TRAM flap developed on genetically obese leptin receptor-negative fa/fa Zucker rats purchased commercially from Harlan Sprague-Dawley, Inc., (Indianapolis, IN).27 The rats weighed 250 to 350 g, were 7 to 8 weeks in age, and of both genders. The rats were housed on a 12–hour light/dark cycle and given food and water ad libitum. All experimental protocols were approved by the Animal Care Use Committee of the Department of Veterans Affairs Medical Center, Bay Pines, Florida.
All operations on the rats were performed under intraperitoneal sodium pentabarbital anesthesia, 35 mg/kg body weight, using aseptic surgical techniques. Once the animals were anesthetized, the abdomens were shaved and the skin prepared with povidone-iodine and 70% isopropyl alcohol. The transverse rectus abdominis musculocutaneous (TRAM) flaps were designed and elevated as originally described by Dunn et al.25 and modified by Khalaf et al.27 The flap, once raised, was placed in an overlay position modified from the position suggested by Clugston et al.26,27 Specifically, a hexagonally shaped template was used to outline the proposed flap, centered on the umbilical dimple. The flap was raised as a unipedicle superiorly based flap attempting to capture the maximal number of periumbilical musculoskeletal perforating vessels.27 Flap elevation included part of the rectus abdominis muscle and anterior rectus sheath encompassing the flap’s vascular pedicle. The remainder of the muscle and the posterior rectus sheath were approximated with absorbable sutures. The flap was overlaid superficial to the approximated fascial layer and the skin was closed with a single layer of continuous nylon sutures insetting the flap to it’s original template shape and dimentions.27
A total of 81 fa/fa Zucker rats were used for the experiments. These were divided into 8 groups of 10 rats each and the extra animal was used as an unmanipulated control from which to obtain biopsies of normal abdominal adipose tissue. The eight groups were as follows: Group 1, control flaps treated with only injections of phosphate buffered saline (PBS) on postoperative days (POD) 0, 1, and 2; Group 2, basic fibroblast growth factor (bFGF) (Scios Corp., Mountain View, CA) flaps treated with injections of 1,000 ng/1.0 mL on POD 0, 1, and 2; Group 3, granulocytic macrophage–colony stimulating factor (GM-CSF) (Shering Plough Research Corp., Kenilworth, NJ) flaps treated with injections of 300 ng/1.0 mL on POD 0, 1, and 2; Group 4, angiotensin II (AII) (Maret Corp., Wayne, PA) flaps treated with injections of 1.0 mg/1.0 mL on POD 0, 1, and 2; Group 5, GM-CSF plus AII flaps treated with injections 300 ng/1.0 mL GM-CSF and 1.0 mg/1.0 mL AII on POD 0, 1, and 2; Group 6, vascular endothelial growth factor, 165 (VEGF-165) (R & D Systems Inc., Minneapolis, MN) flaps treated with injections of 300 ng/1.0 mL on POD 0, 1, and 2; Group 7, vascular endothelial growth factor, 121 (VEGF-121) (R & D Systems Inc., Minneapolis, MN) flaps treated with injections of 300 ng/1.0 mL on POD 0, 1, and 2; and Group 8, platelet-derived endothelial cell growth factor (PD-ECGF) (R & D Systems Inc., Minneapolis, MN) flaps treated with injections of 300 ng/1.0 mL on POD 0, 1, and 2.
All cytokine growth factors were dissolved in PBS. Serum albumin was added to the vehicle for VEGF-165, VEGF-121, and PD-ECGF as recommended by the manufacturers. The doses chosen for each factor was based on recommendations from the providing source and from a review of the literature. After resetting the flaps in the position superficial to the approximated fascia, the cytokines or vehicle control were injected at the four corners of the flap (not at the extreme lateral corners of the hexagon at the midaxillary lines). Then 0.25 mL of the test solution was injected at each site.
Postoperatively, the flaps were monitored for color changes and palpable firmness at 24 hours, 48 hours, and 7 days. On postoperative day 7, the animals were sacrificed and the fat harvested from the flaps, fixed, sectioned, and stained with hematoxylin and eosin (H & E). The histologic sections were evaluated by a blinded pathologist (T.J.S.) to determine the degree of fat necrosis, inflammation, and number of blood vessels. A standard measuring grid was used in all assessments. The representative biopsies were graded on a six-point scale for inflammation and a six-point scale for fat necrosis.27 The inflammation scale ranked the specimens from 0 (no inflammation) to 5 (severe multicellular inflammatory changes in greater than 75% of the specimen). Similarly, fat necrosis was ranked 0 to 5 with 0 being normal healthy fat, and 5 being severe necrosis and liquefaction over 75% of the specimen (Table 1).27 Of the various sections evaluated for each flap, a mode number was determined for the inflammation scale and the fat necrosis scale for each rat.
A Miller disc (normally used for reticulocyte differential counts) was used to count blood vessels. The Miller disc encompasses 0.0484 mm2 at 400 x total magnification (Leitz Diaplan Microscope with MD5 adapter and periplan 10x/20 m eyepieces). The total numbers of blood vessels in 10 randomly selected areas were counted. Any vessels bounded by the Miller disc were included. The total number of counted vessels were multiplied by a factor of 20.66, resulting in vessel density per square millimeter.
A means plus or minus standard deviation inflammation scale rank and fat necrosis scale rank was determined for each treatment group. Comparison of the means of the inflammation scale ranks of each of the cytokine treatment groups to the control group was performed using Dunn’s method for ranked values after a normality designation failed using a Kruskal-Wallis one-way analysis of variance on ranks (P = 0.001). Similarly, the same statistical test was used to compare the means of the fat necrosis scale ranks of each experimental group to the control group. The number of vessels counted in each of the cytokine-treated groups were compared to the number counted in the control group utilizing Bonferroni’s t-test.
Of the 81 fa/fa Zucker rats used in the experiments, 71 survived the entire period and were available for evaluation. Ten rats died or were killed because of cannibalization of the flaps before final biopsy and were eliminated from the histologic results. These rats were fairly evenly distributed among the experimental groups. Two of the rats that died before biopsy could be evaluated clinically for flap color and palpable firmness.
The results for clinical evaluation of the flaps were as follows. The control flaps were uniformly firm to palpation and two flaps demonstrated color change.27 Of the cytokine-treated flaps, all groups contained flaps that remained soft to palpation. Only those treated with bFGF showed firmness in over 50% of the animals. In four of the groups (GM-CSF, GM-CSF+AII, VEGF-165, and PD-ECGF), the flaps were soft throughout in over 85% of the animals (Table 2). In two of the groups (VEGF-165 and PD-ECGF), no color changes occurred throughout the period of observation (Table 2).
Histologically, the control group findings coincided with the clinical findings of firmness. There was a large amount of inflammation and all animals had a large amount of inflammation and all animals had a fat necrosis mode rank of 4 or 5. The mode rankings for both the inflammation scale and the fat necrosis scale were remarkably consistent for the 10 rats.27 Evaluating the control flaps as a group, the mean inflammatory rank was 3.1 + 0.6 and the mean fat necrosis rank was 3.9 + 0.3 (Figs 1 and 2). This compares with no inflammation and no fat necrosis in the normal unmanipulated abdominal adipose tissue (Figs 1 and 2).
Four of the cytokine-treated groups (GM-CSF+AII, PD-ECGF, VEGF-165, VEGF-121) showed significantly less inflammation than the control group (P < .05) (Fig 1). There was a significant difference in all of the cytokine-treated groups vs. the control flaps for the degree of histologic fat necrosis (P < .05) (Fig 2). Figure 3 shows a section of a postoperative day 7 flap treated with VEGF-121 which had the lowest inflammation and fat necrosis scale means compared to a section of a postoperative day 7 control flap.
When comparing new vessel formation in the various treatment groups, all of the flaps showed evidence of increased vascularity, significantly more than the normal unmanipulated adipose tissue (Fig 4). There was not a significant difference between the cytokine-treated groups and the control flap group.
Fat necrosis remains a major challenge for the surgeon and a psychologically devastating problem for the patient. It can present as a hard nodule in a flap used for cancer reconstruction making assessment for local cancer recurrence difficult. Fat necrosis may also change the volume and the contour of the involved tissue, leading to an obvious aesthetic deformity. The involved sites are often painful, tender, and may present as a chronic draining sinus that may take months to resolve. In extreme cases, fat necrosis may result in a partial flap loss requiring a second operation to excise the necrotic fat and recontour the remaining uninvolved tissue.
Because ischemia appears to be the inciting event in the etiology of fat necrosis, it has been postulated that a better arterial supply or venous drainage to flaps susceptible to fat necrosis may lessen the severity of the problem. Surgical manipulation such as delay procedures, use of multiple pedicles, microvascular anastamosis for a portion, or the whole of a flap have been shown to improve vascularity to the flap and decrease both the incidence and degree of fat necrosis. None of these surgical vascular supply augmentations have eliminated the problem of fat necrosis especially in patient conditions of decreased microcirculation such as smoking, obesity, radiation, and so forth.10,35
The experiments described in this paper used angiogenic cytokines in an attempt to decrease or eliminate the ischemia critical for the development of fat necrosis without resorting to an additional operative procedure or microsurgery. Because angiogenesis has been reported to be one of the underlying mechanisms of the delay procedure, using angiogenic cytokines could theoretically attain the desired effect without a two-stage procedure.31 The angiogenic effects of cytokines can be direct, stimulating capillary formation in the absence of inflammation, or indirect, by stimulating neovascularization only through the recruitment of inflammatory cells.33,34 Each of the cytokines chosen have angiogenic effects reported in the literature. Basic FGF (bFGF) has been demonstrated to stimulate capillary endothelial cell proliferation, tube formation, and consequently to induce angiogenesis.34,36,37 Knighton et al.34 state these are indirect effects after stimulation of inflammation. Others have reported a more direct chemotactic and mitogenic effect on endothelial cells resulting in new vessel formation.38,39 GM-CSF stimulates angiogenesis indirectly by being a strong proinflammatory factor that stimulates leukocyte and macrophage activity.40 Angiotensin II facilitates the activation of pre-existing collateral vascular pathways and has direct angiogenic properties.41 By combining GM-CSF and AII in group 5, a biphasic effect of both indirect and direct angiogenesis was sought. Vascular endothelial growth factor (VEGF) is the most powerful direct angiogenic factor that has been identified.42 There are two different varieties of this cytokine, VEGF-165 and VEGF-121. Both of these increase vascular permeability, enhance endothelial cell growth, and promote angiogenesis, as does PD-ECGF.43,44 When PD-ECGF was applied to skin flaps by Hom, et al. to enhance neovascularization, flap survival was improved.45
Each of the angiogenic cytokines injected into the experimental TRAM flaps statistically improved the degree of histologic fat necrosis (Fig. 3). VEGF-121, VEGF-165, PD-ECGF, and the combination GM-CSF and AII had the least degree of fat necrosis and statistically less inflammation (Fig. 1 and Fig. 2). Clinically, the flaps were also soft in three of those four groups (VEGF-165, PD-ECGF, and GM-CSF+AII). Clinically, the GM-CSF alone group was soft, although it did have a medium range degree of inflammation. From these data, it would appear that cytokines that have a direct stimulation of angiogenesis without provoking a great deal of inflammation might be the most useful in preventing fat necrosis.
The data on the average number of blood vessels showed an increase in vessels over normal adipose tissue in all groups. Certainly, the increased numbers seen with all of the cytokines were expected (Fig. 4). One would have expected the VEGF-121 group to show more vessels. However, possibly our method was not sensitive enough to detect small differences. The use of specific histochemistry such as factor 8 staining may have helped detect differences among the cytokines. At first glance, one may wonder why the control flap had an increased number of vessels. However, the control flap is in reality a seven day delayed flap since it was elevated and reset without transfer. As mentioned previously, one of the mechanisms of the delay phenomenon is angiogenesis and neovascularization.31
From these data, it appears that angiogenic cytokines may have a potential use in preventing or decreasing fat necrosis. Since inflammation is an accompanying feature of clinical fat necrosis, it is probably best to explore the use of direct angiogenic factors such as VEGF-165, VEGF-121, or PD-ECGF which do not stimulate inflammation, rather than indirect angiogenic factors such as bFGF, GM-CSF, or AII which stimulate inflammation to a greater degree (Fig. 1). Further experiments are necessary to evaluate different dose responses and different administration routes, e.g. intraarterial into the pedicle. However, if through the exogenous administration of angiogenic factors, the neovascularization process could be accelerated, such cytokine administration might become an alternative to multiple flap medications and/or a two step surgical procedure to decrease the problems that fat necrosis poses to the surgeon and the patient.
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2. Kroll SS, Gherardini G, Martin JE, et al. Fat necrosis in free and pedicled TRAM flaps. Plast Reconstr Surg 102:1502-1507, 1998.
3. Hartrampf CR, Scheflan M, Black PW. Breast reconstruction with a transverse abdominal island flap. Plast Reconstr Surg 69:216-225, 1982.
4. Kroll SS, Evans GRD, Reece GP, et al. Comparison of resource costs of free and conventional TRAM flap breast reconstruction. Plast Reconstr Surg 98:74-77, 1996.
5. Jewell RP, Whitney TM. TRAM fat necrosis in a young surgeon’s practice: Is it experience, technique, or blood flow? Ann Plast Surg 42:424-427, 1999.
6. Schusterman MA, Kroll SS, Weldon ME. Immediate breast reconstruction: Why free TRAM over the conventional TRAM flap. Plast Reconstr Surg 90:255-261, 1992.
7. Eidelman Y, Liebling RW, Buchbinder S, et al. Mammography in the evaluation of masses in breasts reconstructed with TRAM flaps. Ann Plast Surg 41:229-233, 1998.
8. Baldwin BJ, Schusterman MA, Miller MJ, et al. Bilateral breast reconstruction: Conventional versus free TRAM. Plast Reconstr Surg 93:1410-1416, 1994.
9. Hartrampf CR. The transverse abdominal island flap breast reconstruction. A 7-year experience. Clin Plast Surg 15:703-716, 1988.
10. Watterson PA, Bostwick J, Hester TR, et al. TRAM flap anatomy correlated with a 10-year clinical experience with 556 patients. Plast Reconstr Surg 95:1185-1194, 1995.
11. Ishii CH, Bostwick J, Raine TJ, et al. Double-pedicle transverse rectus abdominis myocutaneous flap for unilateral breast and chest wall reconstruction. Plast Reconstr Surg 76:901-907, 1985.
12. Wagner DS, Michelow BJ, Hartrampf CR. Double-pedicle TRAM flap for unilateral breast reconstruction. Plast Reconstr Surg 88:987-997, 1991.
13. Codner MA, Bostwick J, Nahai F, et al. TRAM flap vascular delay for high risk breast reconstruction. Plast Reconstr Surg 96:1615-1622, 1995.
14. Hudson DA. The surgically delayed unipedicled TRAM flap for breast reconstruction. Ann Plast Surg 36:238-242, 1996.
15. Dorion D, Boyd JB, Pang CY. Augmentation of transmidline skin perfusion and viability in transverse rectus abdominis myocutaneous (TRAM) flaps in pigs. Plast Reconstr Surg 88:642-649, 1991.
16. Yamamoto Y, Nohira K, Sugihara T, et al. Superiority of the microvascularly augmented flap: Analysis of 50 transverse rectus abdominis myocutaneous flaps for breast reconstruction. Plast Reconstr Surg 97:79-83, 1996.
17. Yamamoto Y, Nohira K, Shintomi Y, et al. “Turbocharging” the vertical rectus abdominis myocutaneous (turbo-VRAM) flap for breast reconstruction of the extensive chest wall defect. Br J Plast Surg 47:103-107, 1994.
18. Harashina T, Sone K, Inoue T, et al. Augmentation of circulation of pedicled transverse rectus abdominis musculocutaneous flaps by microvascular surgery. Br J Plast Surg 40:367-370, 1987.
19. Grotting JC, Urist MM, Maddox WA, Vasconez LO. Conventional TRAM versus free microsurgical TRAM for immediate breast reconstruction. Plast Reconstr Surg 83:828-841, 1989.
20. Elliott LF, Eskenazi L, Beegle PH, et al. Immediate TRAM flap breast reconstruction: 128 consecutive cases. Plast Reconstr Surg 92:217-227, 1993.
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22. Grotting JC. Immediate breast reconstruction using the free TRAM flap. Clin Plast Surg 21:207-221, 1994.
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27. Khallaf AM, Smith PD, Payne WG, et al. A reliable experimental model of fat necrosis occurring in pedicled flaps utilizing the fa/fa Zucker rat. J Applied Res In press, 2003.
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32. Arranz-Lopez JL, Suarez Nieto C, Barthe Garcia P, Rojo Ortega JM. Evaluation of angiogenesis in delayed skin flaps using monoclonal antibody for the vascular endothelium. Br J Plast Surg 48:479-486, 1995.
33. Phillips GD, Stone AM, Schultz JC, et al. Do growth factors stimulate angiogenesis? A comparison of putative angiogenesis factors. Wounds 9:1-14, 1997.
34. Knighton DR, Phillips GD, Fiegel VD. Wound healing angiogenesis: Indirect stimulation by basic fibroblast growth factor. J Trauma 30:S134-S144, 1990.
35. Williams JK, Bostwick J, Bried JT, et al. TRAM flap breast reconstruction after radiation treatment. Ann Plast Surg 221:756-764, 1995.
36. Moscatelli D, Joseph-Silverstein J, Presta M, Rifkin DB. Multiple forms of an angiogenesis factor: Basic fibroblastic growth factor. Biochemie 70:83-87, 1988.
37. Tsur H, Daniller A, Strauch B. Neovascularization of skin flaps: Route and timing. Plast Reconstr Surg 66:85-90, 1980.
38. Thomas KA., Gimenez-Gallego G. Fibroblast growth factor: Broad-spectrum mitogens with potent mitogenic activity. Trends Biochem Sci 11:81-84, 1986.
39. Ausprunk DH, Folkman J. Migration and proliferation of endothelial cells in preformed and newly formed blood vessels during tumor angiogenesis. Microvasc Res 14:53-65, 1977.
40. Kucukcelebi A, Carp SS, Hayward PG, et al. Granulocyte-macrophage colony stimulating factor reverses the inhibition of wound contraction caused by bacterial contamination. Wounds 4:241, 1992.
41. Fernandez LA, Twicker J, Mead A. Neovascularization produced by angiotensin II. J Lab Clin Med 105:141-145, 1985.
42. Padubidri A, Browne E. Effect of vascular endothelial growth factor (VEGF) on survival of random extension of axial pattern skin flaps in rats. Ann Plast Surg 37:604-611, 1996.
43. Keck PJ, Hauster SD, Krivi G, et al. Vascular permeability factor, an endothelial cell mitogen related to PDGF. Science 246:1309-1312, 1989.
44. Connolly DT, Heuvelman DM, Nelson R, et al. Tumor vascular permeability factor stimulates endothelial cell growth and angiogenesis. J Clin Invest 84:1470-1478, 1989.
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Table 1. Scales for Inflammation and Fat Necrosis in Flap Adipose Tissue
Inflammation Fat Necrosis
0 no inflammation 0 normal adipose tissue
1 minimal inflammation 1 minimal fat necrosis
2 inflammation < 25% of section 2 fat necrosis < 25% of section
3 inflammation > 25% to < 50% of section 3 fat necrosis > 25% to < 50% of section
4 inflammation > 50% to < 75% of section 4 fat necrosis > 50% to < 75% of section
5 inflammation > 75% of section 5 fat necrosis > 75% of section
Table 2. Clinical Evaluation of Flaps
Treatment Palpable Color
Group Firmness Change
Control 10/10 2/10
bFGF 6/10 4/10
GM-CSF 1/10 2/10
AII 3/9 5/9
GM-CSF+AII 1/9 4/9
VEGF-165 0/10 0/10
VEGF-121 3/8 2/8
PD-ECGF 0/10 0/10
Figure 1. Means of seven cytokine-treated flap groups compared to control flap. Normal unmanipulated abdominal adipose tissue placed on far right as a reference showing no inflammation. GM-CSF+AII, PD-ECGF, VEGF-165, VEGF-121 groups showed significantly less inflammation than other groups.
Figure 2. Means of 7 cytokine-treated flap groups compared with control flap. Normal unmanipulated abdominal adipose tissue placed on far right as a reference showing no fat necrosis. All of the cytokine-treated groups showed significantly less fat necrosis compared with the control flaps.
Figure 3. A. Representative section of a control TRAM flap with near complete obliteration of normal fat globules and replacement with inflammatory infiltrates. B. Representative histologic specimen of a TRAM flap treated with VEGF-121 showing increased vascularity and only minimal fat necrosis.
Figure 4. Average number of blood vessels counted in each of the flap groups compared to normal unmanipulated abdominal adipose tissue. All of the flap groups demonstrated increased blood vessels.
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