|Year : 2021 | Volume
| Issue : 5 | Page : 1-8
The effect of adipose stromal vascular fraction on the viability of transverse rectus abdominis myocutaneous flap after abdominoplasty: An experimental study
Erhan Coskun1, Burak Ozkan2, Aysen Terzi3, Eda Ozturan ozer4, Cagri Ahmet Uysal2, Huseyin Borman5, Nilgun Markal Ertas2
1 Department of Plastic Surgery, Esteworld Clinic, Istanbul, Turkey
2 Department of Plastic, Reconstructive and Aesthetic Surgery, Baskent University, Ankara, Turkey
3 Department of Pathology, Baskent University, Ankara, Turkey
4 Department of Biochemistry, Baskent University, Ankara, Turkey
5 Department of Plastic, Reconstructive and Aesthetic Surgery, Bayindir Hospital, Ankara, Turkey
|Date of Submission||22-Mar-2020|
|Date of Acceptance||11-Apr-2020|
|Date of Web Publication||17-Mar-2021|
Dr. Burak Ozkan
Yukarı Bahcelievler Mah 63. Sok 16/17 Cankaya, Ankara
Source of Support: None, Conflict of Interest: None
Introduction: A prior abdominoplasty is considered as an absolute contraindication to transverse rectus abdominis musculocutaneous (TRAM) flap surgery. The aim of this study is to investigate the effect of nonexpanded adipose stromal vascular fraction (ASVF) on the viability of TRAM flap after abdominoplasty. Materials and Methods: Thirty-five male Sprague Dawley rats were divided into five groups. Reverse abdominoplasty model was used in all groups except Group 1. TRAM flap was performed 2 weeks after abdominoplasty in Groups 2 and 4 and 4 weeks after in Groups 3 and 5. ASVF cells were injected during abdominoplasty in Groups 4 and 5. The viable flap area (VFA) percentage and newly formed perforators were assessed. Capillary density and fibrosis gradient and plasma vascular endothelial growth factor (VEGF) levels were measured. Results: The mean VFA to total flap area was measured as 82.90% ± 7.59%, 3.31% ± 3.29%, 9.40% ± 6.18%, 31.92% ± 9.29%, and 64.98% ± 10.95% in Group 1, Group 2, Group 3, Group 4, and Group 5, respectively (P < 0.05). The number of newly formed musculocutaneous perforating arteries was 0.29 ± 0.49, 1.14 ± 0.69, and 2 ± 0.82 for Groups 3, 4, and 5, respectively (P < 0.05). The mean capillary density was 6.86 ± 0.50, 0.67 ± 0.13, 2.79 ± 0.53, 3.71 ± 0.47, and 7.01 ± 0.70 in Groups 1, 2, 3, 4, and 5, respectively (P < 0.05). There was a statistically significant increase between the baseline VEGF values and the second VEGF values in Groups 4 and 5. Conclusions: The study showed that local injection of ASVF increases the viability of TRAM flap after abdominoplasty.
Keywords: Breast reconstruction, stromal vascular fraction, tram flap
|How to cite this article:|
Coskun E, Ozkan B, Terzi A, ozer EO, Uysal CA, Borman H, Ertas NM. The effect of adipose stromal vascular fraction on the viability of transverse rectus abdominis myocutaneous flap after abdominoplasty: An experimental study. Turk J Plast Surg 2021;29, Suppl S1:1-8
|How to cite this URL:|
Coskun E, Ozkan B, Terzi A, ozer EO, Uysal CA, Borman H, Ertas NM. The effect of adipose stromal vascular fraction on the viability of transverse rectus abdominis myocutaneous flap after abdominoplasty: An experimental study. Turk J Plast Surg [serial online] 2021 [cited 2021 Oct 26];29, Suppl S1:1-8. Available from: http://www.turkjplastsurg.org/text.asp?2021/29/5/1/311429
| Introduction|| |
Since Hartrampf et al. introduced the transverse rectus abdominis musculocutaneous (TRAM) flap in 1982; it is one of the most widely used operations for autologous breast reconstruction. The perforator vessels that supply the TRAM flap arise from both the superior and inferior deep epigastric systems. One of the most common complications regarding pedicled TRAM flap is partial tissue loss in the peripheral regions due to low blood flow., Risk factors that compromise this circulation include history of hypertension, diabetes mellitus, obesity, vascular diseases, radiotherapy, smoking, and prior abdominal surgery.,, Classical abdominoplasty is shown to damage the nutritional musculocutaneous perforator arteries of the TRAM flap skin island and considered to be a major contraindication for TRAM flap surgery. Nonetheless, there are several reports of successful TRAM flap harvesting following abdominoplasty in the literature. On the basis of these findings, neovasculogenic factors were used to shorten the time period necessary for safe TRAM flap harvesting following abdominoplasty in several experimental studies.,,
Adipose stromal vascular fraction (ASVF) is easily harvested in high quantities with minimal donor-site morbidity. Stromal vascular fraction is composed of pluripotent stromal cells which are obtained from immediate centrifugation of adipose tissue after collagenase digestion and is promising for angiogenic effects and tissue regeneration.,,,,,
The aim of this study is to evaluate the effect of ASVF on TRAM flap viability following abdominoplasty. According to our literature research, this is the first experimental study that assesses the outcomes of ASVF on TRAM flap harvesting following abdominoplasty.
| Materials and Methods|| |
This study (project number DA14/32) was approved by Baskent University Ethical Committee for Animal Research Studies. Forty-five male Sprague Dawley rats weighing 350–420 g were used for the experiment. Ten rats were operated to attain ASVF and 35 rats were divided into five groups as follows:
- Group 1 (only TRAM flap, n = 7): Only TRAM flap was elevated and nothing else was done
- Group 2 (2 weeks control group, n = 7): Abdominoplasty was performed, and during abdominoplasty, 0.5 ml phosphate-buffered saline (PBS) was injected into the rectus abdominis muscle and subcutaneous plane. TRAM flap was elevated 2 weeks after abdominoplasty
- Group 3 (4 weeks control group, n = 7): Abdominoplasty was performed, and during abdominoplasty, 0.5 ml PBS was injected into the rectus abdominis muscle and subcutaneous plane. TRAM flap was elevated 4 weeks after abdominoplasty
- Group 4 (2 weeks study group, n = 7): Abdominoplasty was performed, and during abdominoplasty, 1.6 × 107 ASVF cells in 0.5 ml PBS were injected into the rectus abdominis muscle and subcutaneous plane. TRAM flap was elevated 2 weeks after abdominoplasty
- Group 5 (4 weeks study group, n = 7): Abdominoplasty was performed, and during abdominoplasty, 1.6 × 107 ASVF cells in 0.5 ml PBS were injected into the rectus abdominis muscle and subcutaneous plane. Four weeks after abdominoplasty, TRAM flap was elevated.
In all groups, a silicon sheet was sutured to the donor site of the flap to prevent neovascularization from the wound bed, next the flaps were sutured back to their novel place.
Isolation and preparation of adipose stromal vascular fraction
Ten rats were anesthetized with intraperitoneally injected 40 mg/kg ketamine hydrochloride (Ketosal; Interhas Co. Ltd., Ankara, Turkey) and 5 mg/kg xylazine hydrochloride (Xylazin Bio; Interhas Co. Ltd., Ankara, Turkey). Inguinal and abdominal regions of the rats were shaved. Inguinal fat pads were excised and washed with PBS (Gibco BRL, Grand Island, NY, USA). In compliance with the protocol established by Ogawa et al., stromal vascular fraction cells were harvested and processed.
1,1'-Dioctadecyl-3, 3, 3',3'-tetramethylindocarbocyanine perchlorate (DiI)
1,1′-Dioctadecyl-3, 3, 3′,3′-tetramethylindocarbocyanine perchlorate (DiI) (Invitrogen; Medsantek, Life Technologies, California, USA) was dissolved in 99% ethanol at a concentration of 25% and stored at −20°C for use. Cells were labeled with fluorescent DiI according to the manufacturer's recommendations; cells in suspension were incubated with DiI at a concentration of 2.5 μg/ml in PBS for 5 min at 4°C as described previously.,
Reverse abdominoplasty and transvers rectus abdominis myocutaneous flap model
Reverse abdominoplasty model described by de Freitas et al. was used in all groups except Group 1 (only TRAM flap performed). A trapezoid shape was marked to define the borders of abdominoplasty dissection. A 5 cm transverse line was drawn 1 cm caudal to the xiphoid process as the superior base. The 2 cm inferior base was drawn 3 cm caudal to the superior base. The superior base was incised. The trapezoid-shaped skin flap was detached in a suprafascial plane in the craniocaudal direction, with dividing the perforating vessels. A skin strip measuring 5 cm × 0.4 cm was excised from the superior base to simulate the skin resected during abdominoplasty [Figure 1]. A strip of aponeurosis measuring 3 cm × 0.5 cm located 0.2 cm to the midline was resected of the right rectus abdominis muscle. Solutions were injected locally to the right rectus abdominis muscle at the resection area of the fascia and subcutaneous plane right above the rectus muscle in a total of 5 injection sites [Figure 2]. ASVF cells were injected in Groups 4 and 5 (2-week and 4-week study groups), whereas only PBS was injected in Groups 2 and 3 (2-week and 4-week control groups).
|Figure 2: Schematic view injection sites of the reverse abdominoplasty model|
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Right inferior epigastric artery pedicled 5 cm × 2.5 cm sized TRAM flap was performed as described by Ozgentas et al. 2 weeks after abdominoplasty in Groups 2 and 4 and 4 weeks after in Groups 3 and 5 [Figure 3].
|Figure 3: Illustration of the transverse rectus abdominis myocutaneous flap model|
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Viable flap area
On the 7th day following TRAM flap surgery, standardized photographs of the flaps were taken by a fixed lens (Minolta AF 50 mm, f1,7) installed digital camera (SONY, DSLR-A350) from a fixed distance (50 cm), under a fixed lighting source for the evaluation of the Viable flap area. Flap viability was calculated using the program ImageJ 1.48 v (National Institutes of Health, 9000 Rockville Pike, Bethesda, Maryland 20892, USA); Java 1.6.0_65 (image processing and analysis in Java) by pixel count.
The number of musculocutaneous perforator arteries
The number of musculocutaneous perforator arteries was counted at two different stages during the study period. The first stage is during the abdominoplasty. A transverse 5 cm long incision was done 1 cm below the sternal notch to initiate abdominoplasty. Meticulous dissection under surgical microscope was carried out to expose the rectus musculocutaneous perforator arteries and all the arteries were marked with painted paper markers [Figure 4]. All the perforators were counted and were ligated using bipolar electrocautery. After seven days we remove the TRAM flaps for we remove the TRAM flaps for tissue examination, we dissected Zone I under surgical microscope to see whether any newly formed musculocutaneous perforators exist [Figure 5].
|Figure 4: Markings of the abdominal perforators while reverse abdominoplasty performing|
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|Figure 5: Investigation of Zone I of tram flap for newly formed musculocutaneous perforators before harvesting of tram flap|
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Five micrometer tissue sections were sliced horizontally comprising all the zones of the skin and the rectus muscle and stained with hematoxylin and eosin. Neovascularization was assessed by measuring the number of capillaries in 20 different fields under × 40 magnification according to the previous studies.
Muscular fibrosis gradient
Five micrometer tissue sections were sliced from the paraffin blocs for Masson's trichrome staining. The damage to the rectus abdominis muscle was evaluated with the fibrosis amount (fibrosis gradient). The fibroblastic activity, fibrosis level, and degeneration level of the myocytes were graded as described previously, (grade 0: normal, grade 1: mild fibrosis, grade 2: moderate fibrosis, and grade 3: severe fibrosis and presence of myocyte degeneration).
DiI-labeled adipose stromal vascular fraction cells in the tissues prompt
Five micrometer tissue sections were sliced from the paraffin blocs and stained only by hematoxylin. DiI is visible at the 565 μm wavelength; hence, DiI-labeled cells in the tissues were identified under a fluorescent microscope applying the assured wavelength. The same fields were photographed under the florescent and light microscope separately, later these photos were merged to designate DiI-labeled stromal vascular fraction (SVF) cells in the tissues as described previously.
Plasma vascular endothelial growth factor levels
In all groups except Group 1, 1 cc of blood samples was taken from the tail veins using a 22 G needle on the days of abdominoplasty (1st day of the study), TRAM flap elevation, and 7 days after TRAM flap elevation. The blood samples taken before the operation on the 1st day were used to determine the basal levels of vascular endothelial growth factor (VEGF). The plasma of the blood sample was collected after centrifugation at 4000 rpm for 10 min. Enzyme-linked immunosorbent assay (ELISA) was performed by Rat VEGF Elisa Kit (RayBiotech, Inc., Norcross GA, USA). Biotin-labeled anti-rat VEGF antibody was incubated with the plasma of the blood sample for 1 h. Then, avidin-labeled peroxidase was added and incubated for 45 min. The addition of the 3,3′, 5, 5′-tetramethylbenzidine substrate of the peroxidase enzyme has yielded blue coloration. After 30 min, the stop solution of the peroxidase terminated the enzymatic reaction and changed the color to yellow. The density of this coloration was measured with the plate reader at 450 nm and the VEGF level was recorded as pg/ml.
The flap viability percentage, the number of existing musculocutaneous perforator arteries before abdominoplasty, the number of newly formed musculocutaneous perforator arteries after abdominoplasty, vascular density, fibrosis gradient, and blood VEGF levels were statistically evaluated between the groups. The data were presented as mean ± standard deviation. The homogeneity of the group variances was checked with Levene test. The compliance of the continuous variables to the normal distribution was controlled with Shapiro–Wilk test. If the pretest results supported the prerequisites, the variables of the two group's results were analyzed with Student's t-test. If the prerequisites were not supported, the variables were analyzed using Mann–Whitney U-test. If the pretest results supported the prerequisites, the variables of the three or more group's results were analyzed using one-way variance analysis. If the prerequisites were not supported, the variables were analyzed using Kruskal–Wallis test. Differences were regarded as statistically significant if P < 0.05. SSPS software (SPSS ver. 17.0, SSPS Inc., Chicago, IL, USA) was used in every evaluation.
| Results|| |
Viable flap area
The mean percentage of viable flap area (VFA) was 82.90% ± 7.59% in Group 1. The mean percentage of VFAs was 3.31% ± 3.29% and 9.40% ± 6.18% in Groups 2 and 3, respectively. The mean percentage of VFAs was 31.92% ± 9.29% and 64.98% ± 10.95% in Groups 4 and 5, respectively [Figure 6]. According to the pairwise comparison results, there were statistically significant differences between the Groups 2 and 4, 3 and 5, and 4 and 5.
|Figure 6: Flap viabilities of each group and flap viability percentage chart is demonstrated|
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Musculocutaneous perforator arteries
The mean number of musculocutaneous perforator arteries arising from both of the rectus abdominis muscles was 7.98 ± 1.10 before abdominoplasty. There was no newly formed musculocutaneous perforating artery in Group 2. The number of newly formed musculocutaneous perforating arteries after abdominoplasty was 0.29 ± 0.49, 1.14 ± 0.69, and 2 ± 0.82 for Groups 3, 4, and 5, respectively. The number of perforating arteries counted after abdominoplasty was significantly lower than the number of perforating arteries counted before abdominoplasty in all the groups. The number of newly formed perforating arteries after abdominoplasty in Group 5 was statistically significantly higher than Groups 2, 3, and 4 [Table 1].
The mean capillary density was 6.86 ± 0.50, 0.67 ± 0.13, 2.79 ± 0.53, 3.71 ± 0.47, and 7.01 ± 0.70 in Groups 1, 2, 3, 4, and 5, respectively [Figure 7]. According to the pairwise comparison results, there were statistically significant differences between Groups 2 and 4 and 3 and 5 [Table 2].
|Figure 7: The mean vascular density of the groups and images under ×40 magnification in H and E-stained specimens|
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Muscular fibrosis gradient
The mean muscular fibrosis gradient was 1.14 ± 0.69, 1.43 ± 1.13, 1.14 ± 1.21, 1.43 ± 0.98, and 1.14 ± 0.90 in Groups 1, 2, 3, 4, and 5, respectively. The difference between the groups in terms of fibrosis gradients was not statistically significant.
DiI-labeled stromal vascular fraction cells in the tissues
It was shown that ASVF cells marked with DiI participated in the vessel wall structure as endothelial cells by immunohistochemical examination under the fluorescence microscopy [Figure 8].
|Figure 8: Endothelial cells with fluorescent reaction at a wavelength of 565n under fluorescent microscope. Adipose stromal vascular fraction cells marked with DiI, participated in the vessel wall structure as endothelial cells|
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Plasma vascular endothelial growth factor levels
The mean plasma levels of VEGF in Group 2 were 15.39 ± 6.94 pg/ml, 24.53 ± 9.25 pg/ml, and 13.57 ± 11.20 pg/ml in days 1, 14, and 21, respectively. In Group 3, the mean values were 13.43 ± 2.97 pg/ml, 17.50 ± 13.79 pg/ml, and 25.27 ± 23.91 pg/ml in days 1, 28, and 35, respectively. In Group 4, the mean values were 14.00 ± 3.87 pg/ml, 60.89 ± 26.50 pg/ml, and 37.74 ± 32.51 pg/ml in days 1, 14, and 21, respectively. In Group 5, the mean values were 12.63 ± 8.20 pg/ml, 38.32 ± 12.35 pg/ml, and 27.56 ± 12.24 pg/ml in days 1, 28, and 35, respectively. There was statistically significant increase between the baseline VEGF values and the second VEGF values in Groups 4 and 5 [Table 3].
|Table 3: Plasma vascular endothelial growth factor levels of the groups are listed|
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| Discussion|| |
TRAM flap is still considered as the one of the standard options for autologous breast reconstruction. Hartrampf et al. developed a scoring system to determine the surgical risk of TRAM flap patients and abdominoplasty story was given a top score. It has been shown that musculocutaneous perforator arteries that feed TRAM flap skin island and abdominal wall tissue are cut during the abdominoplasty. Contrary to common knowledge, there are several reports of successful TRAM flap harvesting after abdominoplasty. Sozer et al. presented successful cases with bipedicled TRAM flaps with no postoperative complications performed 10 years after abdominoplasty. Ribuffo et al. studied the reperfusion of abdominal perforators after abdominoplasty and measured the perforator arteries of 10 patients with a Doppler ultrasonic device before abdominoplasty and 1, 3, and 6 months after abdominoplasty. They emphasize that vertical rectus abdominis muscle (VRAM) flap should be preferred instead of TRAM flap if autologous reconstruction with abdominal tissue is desired after abdominoplasty. However, esthetic results are as important as the reliability of the selected method in breast reconstruction. TRAM flap is known to have superior esthetic results compared to VRAM flap. Thus, studies to increase flap viability are usually emphasized on TRAM flap.,,,
Many studies investigating tissue regeneration have been made based on the ability of stem cells to transform into different cell types. Mesenchymal stem cells have been promising for regenerative effects since the use of bone marrow-derived stem cells for cardiovascular treatment with promising results., Lately, adipose-derived stem cells gained popularity over bone marrow-derived stem cells, as harvesting is less invasive and is associated with low donor-site morbidity. It has also been shown that adipose tissue contains pluripotent cells in a much more dense manner than bone marrow and has the ability to differentiate into different cells same as bone marrow., However, the use of these multipotent cells in clinical practice has been very difficult due to the “good manufacturing practices” protocols. Hence, the ASVF cell utilization has ended the necessity to culture MSC population., SVF cells have the ability to differentiate into various cell lineages such as osteocytes and chondrocytes. We preferred ASVF due to the feasibility of clinical use.
In this study, the effects of ASVF treatment on TRAM flap viability after abdominoplasty were investigated. The same procedures were repeated at different time intervals. These durations have been determined taking into account similar studies to increase flap survival.,, Our results showed that abdominoplasty damages the perforators of TRAM flap and negatively affects the outcomes of TRAM flap in all experimental groups. The short-term and long-term groups threated with ASVF showed significantly higher VFAs compared to their control groups. This result suggested that ASVF increased the viability of TRAM flap raised after abdominoplasty. Although we utilized ASVF in abdominoplasty made TRAM groups, the lowest rate of necrosis were encountered in non-abdominoplasty group (Group 1). When we compared ASVF-injected short-term and long-term groups to each other, we found significantly higher VFA in the long-term group. This result showed the importance of the time interval between abdominoplasty and elevation of the TRAM flap in agreement with the findings of other studies.,,
We counted musculocutaneous perforator arteries during abdominoplasty and only before raising the TRAM flap. There were significantly higher number of perforators in TRAM flap only group than all other groups. We could not show any newly formed perforator artery in Group 2. ASVF-injected short-term and long-term groups showed significantly higher number of perforators compared to their control groups. It was noted that the structure of newly formed perforator arteries was thin and delicate compared to the ones observed during abdominoplasty. If the time interval between abdominoplasty and elevation of TRAM flaps was increased, increase in the calibration and in the numbers of perforator arteries could be seen. This result suggested that ASVF increased the number of newly formed perforator arteries supplying TRAM flap raised after abdominoplasty depending on the time interval between the abdominoplasty and elevation of TRAM flap.
In our study, vascular density of flaps treated with ASVF presented distinct differences when examined under the light microscopy. The number of vessels was significantly greater in ASVF-treated short- and long-term groups compared to their control groups. The results were in agreement with the findings of other similar studies.,
We injected ASVF into the right rectus muscle and the overlying skin. The fibrosis gradient results of each group were similar to each other, indicating that the injection of ASVF did not cause significant damage to myocytes.
The need for VEGF in vasculogenesis and angiogenesis has been well understood. Studies have shown that mesenchymal stem cells induce angiogenesis by increasing VEGF synthesis.,, In our study, ASVF-treated groups were noted to have significantly increased blood VEGF levels. This result might show that ASVF induces the secretion of VEGF and also directs the secretion of VEGF from newly formed endothelial cells.
| Conclusion|| |
ASVF injection increased the number of vessels and viability of TRAM flap performed after abdominoplasty. ASVF cell therapy is a practical treatment modality for clinical use among other cellular therapies. However, controlled clinical studies are still necessary for cellular therapies to become safer and cheaper for clinical use.
Financial support and sponsorship
This study was financially supported by Baskent University Research Department.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 2]
[Table 1], [Table 2], [Table 3]