Available online at www.sciencedirect.com
British Journal of Oral and Maxillofacial Surgery 52 (2014) 432–437
Perforator flaps—how many perforators are necessary to keep a flap alive? Andreas M. Fichter a , Anna Borgmann a , Lucas M. Ritschl a , David A. Mitchell a , Stefan Wagenpfeil b , Ulf Dornseifer a , Klaus-Dietrich Wolff a , Thomas Mücke a,∗ a b
Department of Oral and Maxillofacial Surgery, Technische Universität München, Klinikum Rechts der Isar, Munich 81675, Germany Institute for Medical Biometriy, Epidemiology and Medical Informatics, Universität des Saarlandes, Homburg, Germany
Accepted 15 February 2014 Available online 11 March 2014
Abstract Perforator flaps are becoming increasingly important in reconstructive microsurgery because of their reduced donor-site morbidity. However, one drawback is partial necrosis caused by vasospasm or inconsistency of delicate perforator vessels. In this study we have evaluated the number and capacity of perforator vessels with respect to the size of a flap, and the influence of vascular endothelial growth factor (VEGF) on the capacity of perforators in a standard animal model. We realised an epigastric perforator flap 4 cm × 7 cm in 36 rats. In 3 control groups (n = 6 in each), flaps were raised based on 4, 2, or 1 perforator vessel(s), while all other perforators as well as the epigastric vessels were ligated. In three study groups (n = 6 in each), set up in the same way as the control groups, we also injected a single dose of VEGF into the wound area. After one week, all areas of necrosis were assessed planimetrically. We also evaluated the wounds by laser Doppler flowmetry preoperatively and after one week, and by histological and immunohistochemical examination. An increased number of perforators, together with VEGF, was associated with a significant reduction in the areas of necrosis. This observation was particularly true in flaps based on only one perforator. The inclusion of additional perforators has a more important role in the success of a flap than theoretical models suggest. Proangiogenetic factors may improve the viability of perforator flaps. © 2014 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
Keywords: Perforator flap; Epigastric flap model; Area of necrosis; Laser Doppler flowmetry; Animal model; Vascular endothelial growth factor
Introduction Perforator flaps are based on cutaneous vessels with small diameters that branch off a main pedicle and perforate fascia or muscle to reach the skin.1 These flaps are becoming more and more popular in reconstructive microsurgery because they are versatile and result in little morbidity at the donor ∗ Corresponding author at: Department of Oral and Maxillofacial Surgery, Klinikum Rechts der Isar der Technischen Universität München, Ismaninger Str. 22, 81675 München, Germany. Tel.: +49 8941402921; fax: +49 8941404993. E-mail addresses: [email protected], [email protected] (T. Mücke).
site. The size of a perforator flap is judged by the size and number of perforators, the volume of tissue included, and the effects of vessels inside the flap. One common drawback of perforator flaps is partial necrosis of skin and fat as a result of inconsistency or frailty of perforator vessels, vasospasm, or other risk factors. If the blood supply of a perforator flap is based on more than one perforator, it is generally thought that it will provide greater versatility in design and reduce the risk of failure. We know of few studies that have focused on the influence of the number of perforators on survival of flaps, and most of the work is based on theoretical models.2,3 Without evidence, the planning of the dimensions of a flap and the number of perforators to be included is mainly judged by the surgeon’s experience.
http://dx.doi.org/10.1016/j.bjoms.2014.02.013 0266-4356/© 2014 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
A.M. Fichter et al. / British Journal of Oral and Maxillofacial Surgery 52 (2014) 432–437
The present study was designed to investigate the influence of the number of perforators on the survival of perforatorbased groin flaps in rats using clinical and non-invasive monitoring with laser Doppler flowmetry. We looked at whether the relations between the number of perforators and areas of necrosis on the flap were linear or exponential. We also investigated whether a single preoperative dose of vascular endothelial growth factor (VEGF) had any influence on the survival of the flap.
Materials and methods Experiments were made according to current German regulations and guidelines for animal welfare and the international principles of care of laboratory animals. We used male Wistar rats (200–230 g), and all procedures were done under general anaesthesia (10% ketamine (1 ml/kg body weight) and 2% xylazine (0.25 ml/kg body weight)) and under aseptic conditions. The anaesthetic was given as a single intraperitoneal injection and supplemented by quarter-doses as necessary. Surgical technique The epigastric flap was raised as a perforator flap based on the method described by Strauch and Murray.4 To evaluate the influence of the capacity of perforators, an oversized flap model was selected to detect areas of low blood supply with a critically-perfused composite flap.5 The size of the flap was marked with a standard 4 cm × 7 cm template. Subsequently it was raised completely based on the perforator vessels of the deep inferior epigastric system, while the pedicle of the superficial epigastric system was ligated or cauterised (Fig. 1).6 After the flap had been raised it was sutured back into the wound bed with 6/0 monofilament interrupted sutures (Ethilon® , Ethicon, Norderstedt, Germany).
433
Laser Doppler flowmetry In addition to recording the colour of the flap and the degree of capillary refill, we measured tissue oxygen saturation (SO2 in %), the haemoglobin concentration (Hb in arbitrary units (AU)), blood flow (AU), and velocity (AU) non-invasively using laser spectrophotometry (Lightguide O2C® , Lea Medizintechnik, Giessen, Germany). This technique has been described in detail by others.8,9 Preoperatively we recorded a baseline measurement and compared it with the measurements taken after 7 days of wound healing. All measurements were taken from the central part of the flap and compared with the same area if possible. If the flap had necrosed, measurements were taken from the central part of the remaining tissue. Measurement of necrotic areas Results were documented using a CCD-camera (type COOLPIX 8700, Nikon, Düsseldorf, Germany) mounted in a perpendicular direction to the flap with a tripod. Pictures of the vital and necrotic areas were analysed (Fig. 1), and the total area of the flap and necrotic areas were calculated using NIH Image Software. Histological examination After 7 days the epigastric flaps were harvested after the necrotic area had been measured, and fixed in 4% formalin solution. The samples were then embedded in paraffin, sectioned at 5 m and stained with haematoxylin and eosin for histological evaluation. For immunohistochemical examination an additional set of cross-sections was created as described above, and stained with endothelial cell markers CD 31 (cluster determinant 31) and vWF (von Willebrand factor), respectively. The technique has been described previously.7 Statistical analysis
Experimental groups Thirty-six rats were randomised from a computer-generated list and subdivided into 6 groups of 6 rats each. In the first 3 groups 4, 2, and 1 perforators, respectively, from the medial epigastric system were left intact, and all the others were cauterised and cut. In the other 3 groups, under identical conditions, but directly before the operation, VEGF 20 l (hVEGF165, PeproTech, Hambourg, Germany) was injected into the area of the proposed wound bed.7 The necrotic areas of each flap were measured after 7 days using planimetry. Flaps were then harvested and the rats killed with a lethal injection of pentobarbital 200 mg/kg (Narcoren® , Rhone-Merieux, Laupheim, Germany).
Data were analysed with SPSS (SPSS for Mac, release 19.0.0.1, Armonk NY, IBM Corp). Differences in the area of necrosis between groups were evaluated using the Mann–Whitney U test, which was also used for evaluation of the results of immunohistochemical staining. Analysis was complemented by multiple linear regression analysis to evaluate the prognostic value of the number of perforators and the effect of VEGF on the rate of necrosis. For regression analysis we used method “enter”, because whether VEGF was given or not was independent of the number of perforators, so they were statistically independent variables used as covariates in the regression model. Generalised Estimating Equations Models were used to evaluate the laser spectrophotometry data. Two-tailed probabilities of less than 0.05 were accepted as significant. All observations were independently
434
A.M. Fichter et al. / British Journal of Oral and Maxillofacial Surgery 52 (2014) 432–437
Fig. 1. Epigastric perforator flap and clinical examples of flap necrosis. Left: the fully raised full-thickness epigastric perforator flap with attached subdermal fat based on 4(1–4) perforator vessels; la = linea alba. Right: clinical examples of partial flap necrosis, ranging from 1.3% to 82.7%.
evaluated by two investigators who were unaware of which groups they were studying.
Results All 36 rats survived the postoperative period without complications and tolerated the anaesthesia and operation well. No epigastric flap showed signs of autocannibalism postoperatively. Area of necrosis The influence of the number of perforators and the injection of VEGF on areas of necrosis are summarised in Table 1. The area of necrosis was significantly lower in groups with more perforators, the difference being significant among all groups (Fig. 2, Table 1). When groups with the same number of perforators were compared, injection of VEGF showed significantly less flap necrosis (Table 1); this was most obvious if only one perforator was left intact (p < 0.001). The findings of the multiple linear regression analysis are shown in Table 1. Laser spectrophotometry The results of laser spectrophotometric investigations are shown in Table 2. It detected significant differences in haemoglobin (AU) at day 7 only between groups with 1 perforator with and without VEGF (p = 0.01). No other laser
Fig. 2. Rate of necrosis. Data are presented as box plots. In the control group, the relation between the number of perforators and the rate of necrosis seems to be non-linear, whereas after vascular endothelial growth factor (VEGF) has been given, this relation seems to be almost linear. The trend lines that connect the median values were created using Microsoft Excel® to illustrate the non-linear relation between the number of perforators and the area of necrosis.
A.M. Fichter et al. / British Journal of Oral and Maxillofacial Surgery 52 (2014) 432–437
435
Table 1 Influence of number of perforators and vascular endothelial growth factor (VEGF) on the area of necrosis. Multiple linear regression analysis shows that both number of perforators and administration of VEGF have a highly significant influence on flap survival. Perforators
VEGF
Rate of necrosis
Exact p value
Mean (SD) %
Median (range)
4 4 2 2 1 1
No Yes No Yes No Yes
14.7 (6.3) 5.39 (4.0) 31.39 (6.54) 10.23 (6.34) 72.19 (6.23) 17.71 (6.72)
15.1 (7.1–27.3) 4.24 (1.3–12.7) 31.4 (24.3–41.9) 10.9 (1.1–17.9) 70.0 (65.6–82.7) 20.7 (6.3–22.9)
 coefficient/R2
0.009 0.002
Multiple regression analysis No of perforators VEGF
British Journal of Oral and Maxillofacial Surgery 52 (2014) 432–437
Perforator flaps—how many perforators are necessary to keep a flap alive? Andreas M. Fichter a , Anna Borgmann a , Lucas M. Ritschl a , David A. Mitchell a , Stefan Wagenpfeil b , Ulf Dornseifer a , Klaus-Dietrich Wolff a , Thomas Mücke a,∗ a b
Department of Oral and Maxillofacial Surgery, Technische Universität München, Klinikum Rechts der Isar, Munich 81675, Germany Institute for Medical Biometriy, Epidemiology and Medical Informatics, Universität des Saarlandes, Homburg, Germany
Accepted 15 February 2014 Available online 11 March 2014
Abstract Perforator flaps are becoming increasingly important in reconstructive microsurgery because of their reduced donor-site morbidity. However, one drawback is partial necrosis caused by vasospasm or inconsistency of delicate perforator vessels. In this study we have evaluated the number and capacity of perforator vessels with respect to the size of a flap, and the influence of vascular endothelial growth factor (VEGF) on the capacity of perforators in a standard animal model. We realised an epigastric perforator flap 4 cm × 7 cm in 36 rats. In 3 control groups (n = 6 in each), flaps were raised based on 4, 2, or 1 perforator vessel(s), while all other perforators as well as the epigastric vessels were ligated. In three study groups (n = 6 in each), set up in the same way as the control groups, we also injected a single dose of VEGF into the wound area. After one week, all areas of necrosis were assessed planimetrically. We also evaluated the wounds by laser Doppler flowmetry preoperatively and after one week, and by histological and immunohistochemical examination. An increased number of perforators, together with VEGF, was associated with a significant reduction in the areas of necrosis. This observation was particularly true in flaps based on only one perforator. The inclusion of additional perforators has a more important role in the success of a flap than theoretical models suggest. Proangiogenetic factors may improve the viability of perforator flaps. © 2014 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
Keywords: Perforator flap; Epigastric flap model; Area of necrosis; Laser Doppler flowmetry; Animal model; Vascular endothelial growth factor
Introduction Perforator flaps are based on cutaneous vessels with small diameters that branch off a main pedicle and perforate fascia or muscle to reach the skin.1 These flaps are becoming more and more popular in reconstructive microsurgery because they are versatile and result in little morbidity at the donor ∗ Corresponding author at: Department of Oral and Maxillofacial Surgery, Klinikum Rechts der Isar der Technischen Universität München, Ismaninger Str. 22, 81675 München, Germany. Tel.: +49 8941402921; fax: +49 8941404993. E-mail addresses: [email protected], [email protected] (T. Mücke).
site. The size of a perforator flap is judged by the size and number of perforators, the volume of tissue included, and the effects of vessels inside the flap. One common drawback of perforator flaps is partial necrosis of skin and fat as a result of inconsistency or frailty of perforator vessels, vasospasm, or other risk factors. If the blood supply of a perforator flap is based on more than one perforator, it is generally thought that it will provide greater versatility in design and reduce the risk of failure. We know of few studies that have focused on the influence of the number of perforators on survival of flaps, and most of the work is based on theoretical models.2,3 Without evidence, the planning of the dimensions of a flap and the number of perforators to be included is mainly judged by the surgeon’s experience.
http://dx.doi.org/10.1016/j.bjoms.2014.02.013 0266-4356/© 2014 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
A.M. Fichter et al. / British Journal of Oral and Maxillofacial Surgery 52 (2014) 432–437
The present study was designed to investigate the influence of the number of perforators on the survival of perforatorbased groin flaps in rats using clinical and non-invasive monitoring with laser Doppler flowmetry. We looked at whether the relations between the number of perforators and areas of necrosis on the flap were linear or exponential. We also investigated whether a single preoperative dose of vascular endothelial growth factor (VEGF) had any influence on the survival of the flap.
Materials and methods Experiments were made according to current German regulations and guidelines for animal welfare and the international principles of care of laboratory animals. We used male Wistar rats (200–230 g), and all procedures were done under general anaesthesia (10% ketamine (1 ml/kg body weight) and 2% xylazine (0.25 ml/kg body weight)) and under aseptic conditions. The anaesthetic was given as a single intraperitoneal injection and supplemented by quarter-doses as necessary. Surgical technique The epigastric flap was raised as a perforator flap based on the method described by Strauch and Murray.4 To evaluate the influence of the capacity of perforators, an oversized flap model was selected to detect areas of low blood supply with a critically-perfused composite flap.5 The size of the flap was marked with a standard 4 cm × 7 cm template. Subsequently it was raised completely based on the perforator vessels of the deep inferior epigastric system, while the pedicle of the superficial epigastric system was ligated or cauterised (Fig. 1).6 After the flap had been raised it was sutured back into the wound bed with 6/0 monofilament interrupted sutures (Ethilon® , Ethicon, Norderstedt, Germany).
433
Laser Doppler flowmetry In addition to recording the colour of the flap and the degree of capillary refill, we measured tissue oxygen saturation (SO2 in %), the haemoglobin concentration (Hb in arbitrary units (AU)), blood flow (AU), and velocity (AU) non-invasively using laser spectrophotometry (Lightguide O2C® , Lea Medizintechnik, Giessen, Germany). This technique has been described in detail by others.8,9 Preoperatively we recorded a baseline measurement and compared it with the measurements taken after 7 days of wound healing. All measurements were taken from the central part of the flap and compared with the same area if possible. If the flap had necrosed, measurements were taken from the central part of the remaining tissue. Measurement of necrotic areas Results were documented using a CCD-camera (type COOLPIX 8700, Nikon, Düsseldorf, Germany) mounted in a perpendicular direction to the flap with a tripod. Pictures of the vital and necrotic areas were analysed (Fig. 1), and the total area of the flap and necrotic areas were calculated using NIH Image Software. Histological examination After 7 days the epigastric flaps were harvested after the necrotic area had been measured, and fixed in 4% formalin solution. The samples were then embedded in paraffin, sectioned at 5 m and stained with haematoxylin and eosin for histological evaluation. For immunohistochemical examination an additional set of cross-sections was created as described above, and stained with endothelial cell markers CD 31 (cluster determinant 31) and vWF (von Willebrand factor), respectively. The technique has been described previously.7 Statistical analysis
Experimental groups Thirty-six rats were randomised from a computer-generated list and subdivided into 6 groups of 6 rats each. In the first 3 groups 4, 2, and 1 perforators, respectively, from the medial epigastric system were left intact, and all the others were cauterised and cut. In the other 3 groups, under identical conditions, but directly before the operation, VEGF 20 l (hVEGF165, PeproTech, Hambourg, Germany) was injected into the area of the proposed wound bed.7 The necrotic areas of each flap were measured after 7 days using planimetry. Flaps were then harvested and the rats killed with a lethal injection of pentobarbital 200 mg/kg (Narcoren® , Rhone-Merieux, Laupheim, Germany).
Data were analysed with SPSS (SPSS for Mac, release 19.0.0.1, Armonk NY, IBM Corp). Differences in the area of necrosis between groups were evaluated using the Mann–Whitney U test, which was also used for evaluation of the results of immunohistochemical staining. Analysis was complemented by multiple linear regression analysis to evaluate the prognostic value of the number of perforators and the effect of VEGF on the rate of necrosis. For regression analysis we used method “enter”, because whether VEGF was given or not was independent of the number of perforators, so they were statistically independent variables used as covariates in the regression model. Generalised Estimating Equations Models were used to evaluate the laser spectrophotometry data. Two-tailed probabilities of less than 0.05 were accepted as significant. All observations were independently
434
A.M. Fichter et al. / British Journal of Oral and Maxillofacial Surgery 52 (2014) 432–437
Fig. 1. Epigastric perforator flap and clinical examples of flap necrosis. Left: the fully raised full-thickness epigastric perforator flap with attached subdermal fat based on 4(1–4) perforator vessels; la = linea alba. Right: clinical examples of partial flap necrosis, ranging from 1.3% to 82.7%.
evaluated by two investigators who were unaware of which groups they were studying.
Results All 36 rats survived the postoperative period without complications and tolerated the anaesthesia and operation well. No epigastric flap showed signs of autocannibalism postoperatively. Area of necrosis The influence of the number of perforators and the injection of VEGF on areas of necrosis are summarised in Table 1. The area of necrosis was significantly lower in groups with more perforators, the difference being significant among all groups (Fig. 2, Table 1). When groups with the same number of perforators were compared, injection of VEGF showed significantly less flap necrosis (Table 1); this was most obvious if only one perforator was left intact (p < 0.001). The findings of the multiple linear regression analysis are shown in Table 1. Laser spectrophotometry The results of laser spectrophotometric investigations are shown in Table 2. It detected significant differences in haemoglobin (AU) at day 7 only between groups with 1 perforator with and without VEGF (p = 0.01). No other laser
Fig. 2. Rate of necrosis. Data are presented as box plots. In the control group, the relation between the number of perforators and the rate of necrosis seems to be non-linear, whereas after vascular endothelial growth factor (VEGF) has been given, this relation seems to be almost linear. The trend lines that connect the median values were created using Microsoft Excel® to illustrate the non-linear relation between the number of perforators and the area of necrosis.
A.M. Fichter et al. / British Journal of Oral and Maxillofacial Surgery 52 (2014) 432–437
435
Table 1 Influence of number of perforators and vascular endothelial growth factor (VEGF) on the area of necrosis. Multiple linear regression analysis shows that both number of perforators and administration of VEGF have a highly significant influence on flap survival. Perforators
VEGF
Rate of necrosis
Exact p value
Mean (SD) %
Median (range)
4 4 2 2 1 1
No Yes No Yes No Yes
14.7 (6.3) 5.39 (4.0) 31.39 (6.54) 10.23 (6.34) 72.19 (6.23) 17.71 (6.72)
15.1 (7.1–27.3) 4.24 (1.3–12.7) 31.4 (24.3–41.9) 10.9 (1.1–17.9) 70.0 (65.6–82.7) 20.7 (6.3–22.9)
 coefficient/R2
0.009 0.002
Multiple regression analysis No of perforators VEGF
