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Prefabrication of large fasciocutaneous flaps using an isolated arterialised vein as implanted vascular pedicle
British Journal of Plastic Surgery (2005) 58, 632–639
Prefabrication of large fasciocutaneous flaps using an isolated arterialised vein as implanted vascular pedicle Nguyen T. Hoanga,*, M. Kloeppelb, R. Staudenmaierc, J. Wernerb, E. Biemerb a
Department of Traumatology and Plastic Surgery of the Central Hospital, 108, No. 1 Tran Hung Dao, Hanoi, Vietnam b Department of Plastic Surgery of the University Hospital rechts der Isar, Technical University of Munich, Munich, Germany c ENT Department of the University Hospital of Regensburg, Regensburg, Germany Received 6 February 2004; accepted 11 January 2005
KEYWORDS Venous pedicle; Implantation; Neovascularised flap
Summary Flap prefabrication represents a new trend in microsurgical tissue transfer. Based on the concept of neovascularisation, in Chinchilla Bastard rabbits (nZ40), an isolated venous pedicle dissected from the femoral and saphena magna vein was arterialised by end-to-end anastomosis to the femoral artery at the inguinal ligament. This arterialised venous loop was implanted beneath a random-pattern vascularised abdominal fasciocutaneous flap as large as 8!15 cm2 to investigate the development of neovascularisation at various evaluating times of 4, 8, 12, 16 and 20 days. To prevent neoangiogenesis from occurring between the underlying vascular bed and abdominal flap, a silicone sheet with the corresponding dimension of 8 cm! 15 cm!0.25 mm was placed and fixed on the abdominal wall. The flap viability and the neovascularisation process in the prefabricated abdominal skin flaps were evaluated by macroscopic observation, blood analysis, selective microangiography and histology. The experimental results showed that newly formed vessels originating from the implanted isolated venous pedicle were evident on the angiograms 4 days after pedicle implantation. In the 8- and 12-day groups, newly formed vessels became larger and some were connected to the originally available vasculature in the abdominal fasciocutaneous flaps. In the 20-day group, entire flaps were perfused by the blood flow supplied from the newly implanted venous pedicles through newly formed vessels and their vascular connections. This study indicated that large flap prefabrication can be created by implantation of an isolated arterialised venous pedicle into a random-pattern vascularised fasciocutaneous flap. Twenty days appears to be the minimal length of time required after arterialised
* Corresponding author. Address: HNO-Klinik und Poliklinik der Universitaet Klinikum Regensburg, Franz-Josef-Strauß-Allee 11, D93053 Regensburg, Germany. Tel.: C49 941 944 9460; fax: C49 941 944 9405. E-mail address: [email protected] (N.T. Hoang).
S0007-1226/$ - see front matter q 2005 The British Association of Plastic Surgeons. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.bjps.2005.01.007
Prefabrication of large fasciocutaneous flaps
633
venous pedicle implantation for the maturation of neovascularisation in the prefabricated flap. q 2005 The British Association of Plastic Surgeons. Published by Elsevier Ltd. All rights reserved.
Free tissue transfer has become a very important technique in plastic and reconstructive surgery.1,2 For reconstruction in certain specific areas such as the face or hands, large and thin reliable wellvascularised tissue flaps from donor sites which are similar in functional and cosmetic properties to the original defect structures are always preferred. In practice, however, no such microvascular flaps are available which satisfy all the requirements of clinical reconstruction. Where appropriate donor flaps are not feasible, flap prefabrication through implantation of a vascular pedicle into a suitable area can provide a valuable solution.3–11 Erol et al.12 reported that a free skin graft buried over the femoral artery and fascia could be elevated as a flap based on the femoral vessel. Hirase et al.13 described neovascularised free flaps using vein grafts as pedicles in rat models, and sufficient neovascularisation was achieved to support the free transfer of these flaps. Morrison et al.14 later investigated neovascularisation in experimental rabbit models and reported the successful elevation of prefabricated flaps using different implanted vascular pedicles. In 2000, Atabey et al.15 used the saphenous and the superficial inferior epigastric pedicle as vascular carriers for successful prefabrication of combined composite flaps in rats. Tanaka and co-workers16 reported in 2003 that an arteriovenous shunt loop or a distal ligation of bundles can be used for producing a tissue-engineered prefabricated skin flap. Our early experimental studies in rabbits indicated that arteriovenous pedicles dissected from the femoral and saphena magna bundles can be successfully used as implanted pedicles in prefabricated flaps.17 For clinical applications, it has been our impression that prefabricated flaps seem to be simpler and more effective when the arteriovenous pedicles are replaced by isolated venous pedicles. This study was, therefore, performed in Chinchilla Bastard rabbits to evaluate the potential of using isolated arterialised venous pedicles in large prefabricated abdominal flaps of 8!15 cm2. A further aim of the study was to investigate the developmental stages and the speed of neovascularisation in random-pattern vascularised fasciocutaneous flaps at various time intervals during flap prefabrication.
Materials and methods In this experiment, female adult Chinchilla Bastard rabbits weighing 3800–4500 g were used, which were housed in accordance with the European Directive for the care and use of lab animals (Reg. Obb. AZ 211-2531-38/95). For the operative procedure, the animals were anaesthetised through intramuscular injection of a 0.4 ml mixture of ketaminhydrochloride 40 mg/kg and xylazin 4 mg/kg. Additional injections of 0.2 ml were used as needed to maintain an adequate level of anaesthesia. Intravenous infusions of saline and 30 ml rheomacrodex 10% were administered intraoperatively through an ear vein during all surgical procedures. An intramuscular injection of an antibiotic (baytril 2.5%, 5 mg/kg) was administrated in the opposite thigh just before the procedure, for antimicrobial prophylaxis. The right abdominal region and lower limb were shaved, washed with soap and sterilised with a povidone iodine solution and draped in a strict manner. In all animals, an 8!15 cm2 rectangular skin flap was marked on the abdominal wall (Fig. 1(A)). Skin incisions were made and the abdominal skin flap including the panniculus carnosus were dissected and lifted up. Haemostasis was controlled with a bipolar electrocautery. A silicone sheet (LPI, Perouse, 60540 Bornel, France) measuring 8 cm! 15 cm!0.25 mm was placed on the abdominal wall and fixed with ethilon 3/0 (ethicon). This silicone functioned as a barrier to stop neoangiogenesis from occurring between the underlying vascular bed and the abdominal fasciocutaneous flap. An incision extending from the inguinal ligament to the heel was made along the medial axis of the femoral and saphena magna bundle. Under a 5! magnification, the femoral and saphena magna vein were identified and dissected very carefully away from the artery to create an isolated vein of 12 cm length. Next, this isolated vein was arterialised by end-to-end anastomosis to the femoral artery at the inguinal ligament using 10/0 monofilament nylon suture (Fig. 1(A)). This arterialised venous loop pedicle which exhibited no folding or twisting was turned cranially and implanted at the centre of the abdominal fasciocutaneous flap, beneath the panniculus carnosus and fixed with vicryl suture 8/0
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Figure 1 (A) Preoperative design of flap prefabrication and the isolated arterialised venous pedicle dissected from the femoral and saphena magna vein before implantation underneath the abdominal fasciocutaneous flap. (B) Implantation of an isolated arterialised venous pedicle underneath a random-pattern vascularised abdominal fasciocutaneous flap: intra- and postoperative view.
(Fig. 1(B)). After irrigation of the pedicle with 1% plain lidocain and warmed heparinised saline (heated to 37 8C), all skin incisions were sutured back into the original positions using interrupted vicryl and nylon 3/0 (Fig. 1(B)). All operated animals were intra- and postoperatively injected with an antibiotic (baytril 2.5% 5 mg/kg i.m.), an analgesic drug (temgesic 0.05 mg/kg i.m.) and 500 IU heparin s.c. This procedure was continued twice daily until the fifth postoperative day. The study animals were divided into five groups of six animals each based on the retention time of 4, 8, 12, 16 and 20 days after pedicle implantation. The animal’s general condition, flap viability and the neovascularisation process in the prefabricated abdominal flaps were evaluated according to the following parameters.
Blood analysis
Macroscopic observation
Selective microangiographic evaluation
The colour and hair growing on the flaps, as well as the presence or absence of necrosis or infections during flap prefabrication were observed. On postoperative day 20, prefabricated flaps with the same dimension and design (8!15 cm2) including the lateral basis were reincised while preserving the
Selective microangiography was performed for all animals in the study and control groups. In the study group, animals were heparinised intravenously and the abdominal skin flaps were incised with the same design and dimensions as the preoperative markings while protecting the implanted venous pedicle. The
newly implanted vascular pedicle, elevated as an island flap based on the implanted pedicle and then sutured back into place using vicryl and nylon 3/0. The viability of these elevated flaps was evaluated daily with respect to colour, area of necrosis, hair growing and wound healing at the flap borders over an observation period of 2 weeks.
Chemical and haematological data such as pH value, partial oxygen pressure (PaO2), partial carbon dioxide pressure (PaCO2), white blood cells (WBC), red blood cells (RBC), haemoglobin (HGB), haematocrit (HCT) and mean cell volume (MCV) were evaluated pre- and postoperatively to determine changes in the blood levels before and after pedicle implantation.
Prefabrication of large fasciocutaneous flaps arterialised venous pedicles were freely dissected in the inguinal ligament. The proximal part of the pedicle was cannulated with a polyethylene tube (0.9 mm!25 cm) and the vascular system of the abdominal prefabricated flap was irrigated with heparinised warmed saline (heated to 37 8C) for 20 min. After irrigation, a suspension of micropaque 30% with rheomacrodex 10% in the ratio of 2:1 was injected into the proximal part of the implanted venous pedicle at a constant injection pressure of 110 mmHg for 45 min. When the microangiographic procedure was finished, animals were given a lethal injection of pentobarbital (160 mg/kg). The flaps were excised and pinned on cardboard holders. Microangiography of the flaps was obtained using a Radiofluor machine (120-TORR Philips, Germany) and Kodak X-ray films (Kodak X:50, France). Standard quantification of neovascularisation in the flaps was performed using an integral line plate with 25 lines for each 15 cm flap length. Counting the vessels was done separately for each flap in the study and control group.
635 jugular vein. After infusion, a suspension of micropaque 30%–rheomacrodex 10% in the ratio of 2:1 was infused into the carotid artery at a constant injection pressure of 110 mmHg for 5 h. After the flaps were excised, all evaluation procedures for microangiography and histology were carried out following exactly the same steps as used for the study group.
Results Thirty-seven rabbits were operated on in the study group, however, seven rabbits died either intraoperatively due to an anaesthetic overdose or postoperatively for unknown reasons. The remaining 30 study animals were randomly divided into five groups of six animals each (six flaps) corresponding to the various retention times of 4, 8, 12, 16 and 20 days after pedicle implantation. Three rabbits (six flaps) were operated on in the control group.
Macroscopic observation Histological evaluation In all groups, neovascularisation in the prefabricated flaps was evaluated by histology. After microangiography, all flaps were immediately immersed in 10% formaline for at least 3 days. Slices were cut perpendicularly to the long axis of the vascular pedicle at the proximal, middle and distal part of the flap, embedded in parafin and stained with haematoxylin eosin (HE), Elastica van Gerson (Evg) and Elastica Ladewig (Eldg). The morphological structures and all vessels perfused by micropaque were carefully observed and counted. Histological quantification was performed at 5! magnification using a 1 cm 2 eyepiece graticule under the light microscope.
Control group To determine the original vasculature in the abdominal skin flap, three rabbits (six flaps) were operated on in the control group. All the incisions were carried out in the same manner as in the study group, but there was no venous pedicle implantation. For microangiography, the control animals were heparinised intravenously and put under general anaesthesia. Plastic intravenous cannulas (0.5!0.9 mm2) were inserted into the carotid artery and jugular vein. Two thousand millilitres of heparinised saline heated to 38 8C was infused into the carotid artery at a standard pressure of 110 mmHg, then allowed to drain through the
In the study group, all abdominal flaps showed no specific findings during flap prefabrication. Necrosis and infections were not observed in any flap. Flap elevation as an island flap was demonstrated in two rabbits of the 20-day group. These elevated flaps showed no necrosis with inconspicuous wound healing and normal hair growth on the flaps similar to that seen in other nonoperated regions. A new fibrovascular connective tissue capsule underlying the flap could be identified in all flaps in the study group. With increased retention time of the pedicle, this fibrovascular capsule became thicker. As a result of this capsule, the arterialised venous pedicle and the abdominal fasciocutaneous flap became integrated, such that the implanted pedicles lay within the prefabricated flap.
Blood analysis A slight anaemia due to intraoperative blood loss was observed postoperatively in all animals of the study group as well as a decrease in haematological data such as WBC, RBC, HGB, HCT and MCV. There were no changes in the pre- and postoperative chemical data.
Selective microangiographic findings The results of vessel quantification from angiograms are presented in Table 1. In the 4-day group, a small number of newly formed vessels originating
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Table 1 Number of vessels counted from angiograms in the 4-, 8-, 12-, 16- and 20-day groups compared to the control group Group
Group Study group
4 days 8 days 12 days 16 days 20 days
Control group
A.N.o.V.
(%)
SD
A.N.o.V.
(%)
SD
2.6 84.5 107.9 175.13 252.34
1.7 35.5 44.1 73.28 105.58
G0.9 G11.8 G14.8 G19.1 G21.35
238.83
100
G5.82
A.N.o.V., average number of vessels; SD, standard deviation; %, percentage of counted vessels.
from the implanted pedicles could be identified (Fig. 2(B)). Eight days after pedicle implantation, the newly formed vessels were larger, more visible and some were connected to the originally available vasculature of the flap (Fig. 2(B)). In the 12-day group, newly formed vessels were more dense and exhibited wider-spread communication with the available vessel system. The vessel quantity in this group had increased to 44.81% compared to the control group (100%). By the 16-day group, newly formed vessels and their vascular connections had continuously progressed, resulting in a variety of different vessel sizes present within the flaps. The vessel quantity in this group reached a value of 73.28%. The 20-day group showed an even higher quantity and density in the vessel network with innumerable vascular communications and the vessel quantity increasing to 105.58% compared to the control group (Fig. 2(C) and (D)). At this point in the neovascular development, the entire prefabricated flaps were receiving blood flow supplied from the newly implanted venous pedicle.
Histological findings All the prefabricated flaps were vital and morphologically unconspicious with normal epidermis, hair follicles, connective tissue and blood vessels. In the 8-, 12-, 16- and 20-day groups, the flaps were slightly thickened due to the development of the new fibrovascular connective tissue capsules underlying the flaps (Fig. 3). This capsule developed from elastic collagen fibres with a rich capillary network. In the 8-day and 12-day group, the newly formed vessels had penetrated into the subdermal tissue of the flaps, especially around the implanted pedicle. In the 20-day group, the contrast medium spread out through newly formed vessels over the entire vascular system of the prefabricated abdominal flaps (Fig. 3). The
histological quantification was similar to that of the microangiographic findings.
Discussion Neovascularisation is a natural and indispensable part of wound healing. Although the precise mechanisms of neovascularisation are not well understood, it can be selectively combined with vascular implantation and microsurgical techniques to create customised neovascularised free flaps.15–17 Prefabricated free flaps have been investigated by different authors.18–27 Erol and Spira12 used omentum majus as sources of vascularity for skin grafts. In rat and rabbit models, Hirase et al.13 reported several types of neovascularised free flaps including muscle, bone, cutaneous, myocutaneous, fat and perichondrial flaps. Morrison et al.14 showed in an experimental study that neovascularisation began within 2 weeks and achieved maximum development by 8–12 weeks, irrespective of which vascular bundle, arteriovenous loop or artery was implanted below the elevated flap. Nichter et al.27 indicated from an experimental study on arterialised venous flaps in rabbits that arterial inflow is needed for predictable survival of composite tissue through the venous tree or the eventual ingrowth of recipient vessels into the flap by way of the delay phenomena. Based on the concept of a ‘flow-through’ vein, Takato et al.28 investigated the viability of prefabricated total venous perfusion flaps using the left epigastric vein in a rabbit model. They concluded that reproducible survival of prefabricated flaps can be obtained when the flap was elevated more than 3 weeks after prefabrication. Karatas and co-workers29 investigated the prefabrication of composite arteriovenous flaps with implantation of an autologous graft (cartilage) or an alloplastic material (porous
Prefabrication of large fasciocutaneous flaps
637 polyethylene) in rabbits, and showed that arteriovenous perfusion can nourish a prefabricated flap containing an implanted material (autologous or alloplastic) and that 2-week delayed composite flaps have a similar survival rate to delayed prefabricated conventional axial flaps. Applying a different model as these investigators, we have created prefabricated flaps in Chinchilla Bastard rabbits as large as 8!15 cm2 using an isolated vein dissected from the femoral and saphena magna vein, arterialised by end-to-end anastomosis to the femoral artery at the inguinal ligament, as an implanted vascular pedicle. A silicone sheet with the corresponding dimension of 8 cm!15 cm!0.25 mm was placed on the abdominal wall to prevent angiogenesis from occurring between the underlying vascular bed and the abdominal flap.30 The selective microangiographic and histological results showed that newly formed vessels arising from the implanted pedicles were already evident 4 days after pedicle implantation and silicone insertion. With increased retention time of the pedicle, they began communicating with the originally available vasculature in the abdominal prefabricated flap to establish a newly neovascularised-rich system. Twenty days after implantation, the results of macroscopic observation, microangiographic and histological findings showed that the entire abdominal fasciocutaneous flap was well perfused by the blood flow supplied from the newly implanted arterialised venous pedicle through newly formed vessels and their vascular connections. At this point in neovascular development, the vessel quantity on microangiograms had increased to 105.58% compared to the control group. The newly neovascularised system in the flaps consisted of the implanted pedicle, newly formed vessels, the originally available vasculature and their rich vascular connections. Our
Figure 2 (A) Selective microangiography of the 4-day group. (B) Selective microangiography of the 8-day group. Newly formed vessels arising from the implanted pedicle were connected with the originally vasculature in the abdominal fasciocutaneous flap. (C) Selective microangiography of the 20-day group showed the newly neovascularised system in the prefabricated flap. The entire vascular system of the flap was perfused by the blood flow supplied from the newly implanted arterialised venous pedicle. (D) Microangiography of the control group shows the originally available vasculature in the abdominal fasciocutaneous flap.
Figure 3 Histologic specimen of the 20-day group (EvG—stain). The contrast medium spread out through newly formed vessels into the entire vasculature of the flap.
638 observations were similar to those of Itoh,31 who showed in an experimental model in rats, that newly formed vessels were generated not only from the implanted vascular pedicle, but also from the original vessels of the flap. Most of the vascular proliferation occurred within the first week. An important question related to the clinical application of prefabricated flaps using isolated arterialised venous pedicles, is how long it takes for the matured neovacularisation process to occur in the flap after pedicle implantation. In the present study, selective microangiography demonstrated that 20 days after pedicle implantation, the entire flap was perfused by the blood flow supplied from the implanted pedicles. Histological evaluations of the 20-day group showed a normal skin subdermal architecture with the micropaque spreading out over the entire vascular system in the flaps. Our results indicate that 20 days is the minimal length of time required after pedicle implantation for maturation of neovascularisation in prefabricated flaps using silicone sheets and isolated arterialised venous pedicles. The influence of the panniculus carnosus on the increase in neovascularsation in prefabricated flaps was reported by many authors.4,16,18,31 The paniculus carnosus, with its rich capillary vascular network, can play an important role in establishing vascular connections in the flaps. For clinical applications, the fascia underlying the flap must be protected to stimulate the neovascularisation processes in these flaps. The technique of flap prefabrication using isolated arterialised venous pedicles for clinical applications in defect reconstruction is quite promising. This procedure allows surgeons to select implanted vascular pedicles very easily and safely based on the abundant venous system of the human body. Applying the principles of the prefabricated flap, custom-made neovascularised flaps using isolated arterialised venous pedicles can be created in desired regions of similar colour, thickness and dimension regardless of its native vascular anatomy. The technique results in a reduction in functional morbidity and more aesthetic donor defects. Disadvantages of the method are the need for a two-stage operation and the scar in the venous pedicle donor site. Flap prefabrication by means of pedicle implantation into a random-pattern vascularised tissue area represents a new and powerful tool for reconstructive surgeons. The technique allows the creation of reliable axial well-revascularised flaps, which satisfy specific requirements for defect reconstruction. Based on this study, it can be concluded that an isolated arterialised venous
N.T. Hoang et al. pedicle can be used as an implanted vascular pedicle in a prefabricated flap. Twenty days is the minimal period of time required after pedicle implantation for the maturation of the neovascularisation in prefabricated flaps using silicone sheets and isolated arterialised venous pedicles. These experimental results have shown the promising potential of isolated venous pedicles for clinical applications.
Acknowledgements This work was financially supported by the DFG (Deutsche Forschungsgemeischaft). We also wish to thank Dr K. Herfeldt and Dr O. Petrowicz—Institute for Experimental Surgery of the University Hospital rechts der Isar of the Technical University of Munich, Germany for their help and advice in performing the histological images and statistical analysis.
References 1. Biemer E, Duspiva W. Reconstructive microvascular surgery. Berlin: Springer; 1982. p. 3–29; see also 105–42. 2. Geishauser M, Schwachz M. Freie mikrovaskulaere und axialgestielte. Lappen: Venoese Lappen; 1995. p. 218–20. 3. Schechter GL, Biller HF, Ogura JH. Revascularized skin flaps: a new concept in transfer of skin flaps. The Laryngoscope 1969;79:1647–65. 4. Freedman AM, Meland NB. Arteriovenous shunt in free vascularized tissue transfer for extremity reconstruction. Ann Plast Surg 1989;23:123–8. 5. Germann G, Pelzer M, Sauerbier M. Vorfabrizierte Lappenplastiken (‘prefabricated flap’) Ein neues rekonstruktives Konzept. Orthopaede 1998;27:451–6. 6. Pribaz JJ, Fine N, Orgill DP. Flap prefabrication in the head and neck: a 10-year experience. Plast Reconstr Surg 1999; 103:808–20. 7. Kimura N, Hasumi T, Satoh K. Prefabricated thin flap using the transversalis fascia as a carrier. Plast Reconstr Surg 2001;108:1972–80. 8. Sakai S. Reconstruction of an amputated fingertip by a prefabricated free volar forearm flap. Br J Plast Surg 2002; 55:523–6. 9. Safak T, Akyurek M, Ozcan G, Kecik A, Aydin M. Osteocutaneous flap prefabrication based on the principle of vascular introduction: an experimental and clinical study. Plast Reconstr Surg 2000;105:1304–13. ¨ zerdem O ¨ R, Anlatici R, Sen O, Yildirim T, Bircan S, Aydin M. 10. O Prefabricated galeal flap based on superficial temporal and posterior auricular vessels. Plast Reconstr Surg 2003;111: 2166–75. 11. Rohner D, Jaquiery C, Kunz C, Bucher P, Maas H, Hammer B. Maxillofacial reconstruction with prefabricated osseous free flaps: a 3-year experience with 24 patients. Plast Reconstr Surg 2003;112:748–57.
Prefabrication of large fasciocutaneous flaps 12. Erol O, Spira M. Omentum island skin graft flap. Surgical Forum 1978;29:594–6. 13. Hirase Y, Valauri FA, Buncke HJ, Newlin LJ. Customized prefabricated neovascularized free flaps. Microsurgery 1987;8:218–24. 14. Morrison WA, Dvir E, Doi K, Hurley MJ, O’Brien B. Prefabrication of thin transferable axial-pattern skin flaps: an experimental study in rabbits. Br J Plast Surg 1990;43: 645–54. 15. Atabey A, Mc Carthy E, Manson P, Kolk CAV. Prefabrication of combined composite (chimeric) flap in rats. Ann Plast Surg 2000;45:484–90. 16. Tanaka Y, Sung KC, Tsutsumi A, Ohba S, Ueda K, Morrison WA. Tissue engineering skin flaps: which vascular carrier, arteriovenous shunt loop or arteriovenous bundle, has more petential for angiogenesis and tissue generation? Plast Reconstr Surg 2003;112:1626–44. 17. Nguyen T. Hoang. Die Neovaskularisation im praeformierten Gewebelappen in Abhaengigkeit von arteriovenoesen Blutfluss des implantierten Gefaesstieles. Dissertation TU Munich; 1997, p. 1–127. 18. Shen Z, Wang S, Cheng X, Lu J, Yin D, Sun Y. Greater omentum-cutaneous axial flap: a method to create transferable skin flap. Chin Med J 1981;94:718–22. 19. Erk Y, Rose FA, Spira M. Vascular augmentation of skin and musculotaneous flaps. Ann Plast Surg 1983;10:341–8. 20. Kwan CT, Khouri RK, Keuk SS, Shaw WW. The fasciovascular pedicle for revascularisation of other tissues. Ann Plast Surg 1991;26:149–55. 21. Maitz PKM, Pribaz JJ, Duffy FJ, Hergrueter CA. The value of the delay phenomenon in flap prefabrication: an experimental study in rabbits. Br J Plast Surg 1994;47:149–54.
639 22. Adams WP, Griffin JR, Friedman RM, Rohricht RJ, Robinson JB. The myoadipose flap: a new composite. Plast Reconstr Surg 1998;102:735–40. 23. Hickey MJ, Wilson J, Hurley JV, Morrisson WA. Mode of vascularisation of control and basic fibroblast growth factorstimulated prefabricated skin flaps. Plast Reconstr Surg 1998;101:1296–304. 24. Alm MI, Asahina I, Seto I, Oda M, Enotomo S. Prefabricated vascularized bone flap: a tissue transformation technique for bone reconstruction. Plast Reconstr Surg 2001;108:952–8. 25. Hong JP, Lee HB, Chung YK, Kim SW, Tark KC. Coverage of difficult wounds around the knee joint with prefabricated, distally based sartorious muscle flaps. Ann Plast Surg 2003; 50:484–90. 26. Macleod TM, Williams G, Sanders R, Green CJ. Prefabricated skin flaps in rat model based on dermal replacement matrix Permacole. Br J Plast Surg 2003;56:775–83. 27. Nichter LS, Haines PC. Arterialized venous perfusion of composite tissue. Am J Surg 1985;150:191–6. 28. Takato T, Komuro Y, Yonehara H, Zucker RM. Prefabricated venous flap: an experimental study in rabbits. Br J Plast Surg 1993;46:122–6. ¨ , Atabey A, Demirdo 29. Karatas O ¨ver C, Barutcu A. Delayed prefabricated arterial composite venous flaps: an experimental study in rabbits. Ann Plast Surg 2000;44:44–52. 30. Braley SA. The use of silicones in plastic surgery. Plast Reconst Surg 1973;51:280–8. 31. Itoh Y. An experimental study of prefabricated flaps using silicone sheets, with reference to the vascular patternization process. Ann Plast Surg 1992;28:140–6.
Prefabrication of large fasciocutaneous flaps using an isolated arterialised vein as implanted vascular pedicle Nguyen T. Hoanga,*, M. Kloeppelb, R. Staudenmaierc, J. Wernerb, E. Biemerb a
Department of Traumatology and Plastic Surgery of the Central Hospital, 108, No. 1 Tran Hung Dao, Hanoi, Vietnam b Department of Plastic Surgery of the University Hospital rechts der Isar, Technical University of Munich, Munich, Germany c ENT Department of the University Hospital of Regensburg, Regensburg, Germany Received 6 February 2004; accepted 11 January 2005
KEYWORDS Venous pedicle; Implantation; Neovascularised flap
Summary Flap prefabrication represents a new trend in microsurgical tissue transfer. Based on the concept of neovascularisation, in Chinchilla Bastard rabbits (nZ40), an isolated venous pedicle dissected from the femoral and saphena magna vein was arterialised by end-to-end anastomosis to the femoral artery at the inguinal ligament. This arterialised venous loop was implanted beneath a random-pattern vascularised abdominal fasciocutaneous flap as large as 8!15 cm2 to investigate the development of neovascularisation at various evaluating times of 4, 8, 12, 16 and 20 days. To prevent neoangiogenesis from occurring between the underlying vascular bed and abdominal flap, a silicone sheet with the corresponding dimension of 8 cm! 15 cm!0.25 mm was placed and fixed on the abdominal wall. The flap viability and the neovascularisation process in the prefabricated abdominal skin flaps were evaluated by macroscopic observation, blood analysis, selective microangiography and histology. The experimental results showed that newly formed vessels originating from the implanted isolated venous pedicle were evident on the angiograms 4 days after pedicle implantation. In the 8- and 12-day groups, newly formed vessels became larger and some were connected to the originally available vasculature in the abdominal fasciocutaneous flaps. In the 20-day group, entire flaps were perfused by the blood flow supplied from the newly implanted venous pedicles through newly formed vessels and their vascular connections. This study indicated that large flap prefabrication can be created by implantation of an isolated arterialised venous pedicle into a random-pattern vascularised fasciocutaneous flap. Twenty days appears to be the minimal length of time required after arterialised
* Corresponding author. Address: HNO-Klinik und Poliklinik der Universitaet Klinikum Regensburg, Franz-Josef-Strauß-Allee 11, D93053 Regensburg, Germany. Tel.: C49 941 944 9460; fax: C49 941 944 9405. E-mail address: [email protected] (N.T. Hoang).
S0007-1226/$ - see front matter q 2005 The British Association of Plastic Surgeons. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.bjps.2005.01.007
Prefabrication of large fasciocutaneous flaps
633
venous pedicle implantation for the maturation of neovascularisation in the prefabricated flap. q 2005 The British Association of Plastic Surgeons. Published by Elsevier Ltd. All rights reserved.
Free tissue transfer has become a very important technique in plastic and reconstructive surgery.1,2 For reconstruction in certain specific areas such as the face or hands, large and thin reliable wellvascularised tissue flaps from donor sites which are similar in functional and cosmetic properties to the original defect structures are always preferred. In practice, however, no such microvascular flaps are available which satisfy all the requirements of clinical reconstruction. Where appropriate donor flaps are not feasible, flap prefabrication through implantation of a vascular pedicle into a suitable area can provide a valuable solution.3–11 Erol et al.12 reported that a free skin graft buried over the femoral artery and fascia could be elevated as a flap based on the femoral vessel. Hirase et al.13 described neovascularised free flaps using vein grafts as pedicles in rat models, and sufficient neovascularisation was achieved to support the free transfer of these flaps. Morrison et al.14 later investigated neovascularisation in experimental rabbit models and reported the successful elevation of prefabricated flaps using different implanted vascular pedicles. In 2000, Atabey et al.15 used the saphenous and the superficial inferior epigastric pedicle as vascular carriers for successful prefabrication of combined composite flaps in rats. Tanaka and co-workers16 reported in 2003 that an arteriovenous shunt loop or a distal ligation of bundles can be used for producing a tissue-engineered prefabricated skin flap. Our early experimental studies in rabbits indicated that arteriovenous pedicles dissected from the femoral and saphena magna bundles can be successfully used as implanted pedicles in prefabricated flaps.17 For clinical applications, it has been our impression that prefabricated flaps seem to be simpler and more effective when the arteriovenous pedicles are replaced by isolated venous pedicles. This study was, therefore, performed in Chinchilla Bastard rabbits to evaluate the potential of using isolated arterialised venous pedicles in large prefabricated abdominal flaps of 8!15 cm2. A further aim of the study was to investigate the developmental stages and the speed of neovascularisation in random-pattern vascularised fasciocutaneous flaps at various time intervals during flap prefabrication.
Materials and methods In this experiment, female adult Chinchilla Bastard rabbits weighing 3800–4500 g were used, which were housed in accordance with the European Directive for the care and use of lab animals (Reg. Obb. AZ 211-2531-38/95). For the operative procedure, the animals were anaesthetised through intramuscular injection of a 0.4 ml mixture of ketaminhydrochloride 40 mg/kg and xylazin 4 mg/kg. Additional injections of 0.2 ml were used as needed to maintain an adequate level of anaesthesia. Intravenous infusions of saline and 30 ml rheomacrodex 10% were administered intraoperatively through an ear vein during all surgical procedures. An intramuscular injection of an antibiotic (baytril 2.5%, 5 mg/kg) was administrated in the opposite thigh just before the procedure, for antimicrobial prophylaxis. The right abdominal region and lower limb were shaved, washed with soap and sterilised with a povidone iodine solution and draped in a strict manner. In all animals, an 8!15 cm2 rectangular skin flap was marked on the abdominal wall (Fig. 1(A)). Skin incisions were made and the abdominal skin flap including the panniculus carnosus were dissected and lifted up. Haemostasis was controlled with a bipolar electrocautery. A silicone sheet (LPI, Perouse, 60540 Bornel, France) measuring 8 cm! 15 cm!0.25 mm was placed on the abdominal wall and fixed with ethilon 3/0 (ethicon). This silicone functioned as a barrier to stop neoangiogenesis from occurring between the underlying vascular bed and the abdominal fasciocutaneous flap. An incision extending from the inguinal ligament to the heel was made along the medial axis of the femoral and saphena magna bundle. Under a 5! magnification, the femoral and saphena magna vein were identified and dissected very carefully away from the artery to create an isolated vein of 12 cm length. Next, this isolated vein was arterialised by end-to-end anastomosis to the femoral artery at the inguinal ligament using 10/0 monofilament nylon suture (Fig. 1(A)). This arterialised venous loop pedicle which exhibited no folding or twisting was turned cranially and implanted at the centre of the abdominal fasciocutaneous flap, beneath the panniculus carnosus and fixed with vicryl suture 8/0
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Figure 1 (A) Preoperative design of flap prefabrication and the isolated arterialised venous pedicle dissected from the femoral and saphena magna vein before implantation underneath the abdominal fasciocutaneous flap. (B) Implantation of an isolated arterialised venous pedicle underneath a random-pattern vascularised abdominal fasciocutaneous flap: intra- and postoperative view.
(Fig. 1(B)). After irrigation of the pedicle with 1% plain lidocain and warmed heparinised saline (heated to 37 8C), all skin incisions were sutured back into the original positions using interrupted vicryl and nylon 3/0 (Fig. 1(B)). All operated animals were intra- and postoperatively injected with an antibiotic (baytril 2.5% 5 mg/kg i.m.), an analgesic drug (temgesic 0.05 mg/kg i.m.) and 500 IU heparin s.c. This procedure was continued twice daily until the fifth postoperative day. The study animals were divided into five groups of six animals each based on the retention time of 4, 8, 12, 16 and 20 days after pedicle implantation. The animal’s general condition, flap viability and the neovascularisation process in the prefabricated abdominal flaps were evaluated according to the following parameters.
Blood analysis
Macroscopic observation
Selective microangiographic evaluation
The colour and hair growing on the flaps, as well as the presence or absence of necrosis or infections during flap prefabrication were observed. On postoperative day 20, prefabricated flaps with the same dimension and design (8!15 cm2) including the lateral basis were reincised while preserving the
Selective microangiography was performed for all animals in the study and control groups. In the study group, animals were heparinised intravenously and the abdominal skin flaps were incised with the same design and dimensions as the preoperative markings while protecting the implanted venous pedicle. The
newly implanted vascular pedicle, elevated as an island flap based on the implanted pedicle and then sutured back into place using vicryl and nylon 3/0. The viability of these elevated flaps was evaluated daily with respect to colour, area of necrosis, hair growing and wound healing at the flap borders over an observation period of 2 weeks.
Chemical and haematological data such as pH value, partial oxygen pressure (PaO2), partial carbon dioxide pressure (PaCO2), white blood cells (WBC), red blood cells (RBC), haemoglobin (HGB), haematocrit (HCT) and mean cell volume (MCV) were evaluated pre- and postoperatively to determine changes in the blood levels before and after pedicle implantation.
Prefabrication of large fasciocutaneous flaps arterialised venous pedicles were freely dissected in the inguinal ligament. The proximal part of the pedicle was cannulated with a polyethylene tube (0.9 mm!25 cm) and the vascular system of the abdominal prefabricated flap was irrigated with heparinised warmed saline (heated to 37 8C) for 20 min. After irrigation, a suspension of micropaque 30% with rheomacrodex 10% in the ratio of 2:1 was injected into the proximal part of the implanted venous pedicle at a constant injection pressure of 110 mmHg for 45 min. When the microangiographic procedure was finished, animals were given a lethal injection of pentobarbital (160 mg/kg). The flaps were excised and pinned on cardboard holders. Microangiography of the flaps was obtained using a Radiofluor machine (120-TORR Philips, Germany) and Kodak X-ray films (Kodak X:50, France). Standard quantification of neovascularisation in the flaps was performed using an integral line plate with 25 lines for each 15 cm flap length. Counting the vessels was done separately for each flap in the study and control group.
635 jugular vein. After infusion, a suspension of micropaque 30%–rheomacrodex 10% in the ratio of 2:1 was infused into the carotid artery at a constant injection pressure of 110 mmHg for 5 h. After the flaps were excised, all evaluation procedures for microangiography and histology were carried out following exactly the same steps as used for the study group.
Results Thirty-seven rabbits were operated on in the study group, however, seven rabbits died either intraoperatively due to an anaesthetic overdose or postoperatively for unknown reasons. The remaining 30 study animals were randomly divided into five groups of six animals each (six flaps) corresponding to the various retention times of 4, 8, 12, 16 and 20 days after pedicle implantation. Three rabbits (six flaps) were operated on in the control group.
Macroscopic observation Histological evaluation In all groups, neovascularisation in the prefabricated flaps was evaluated by histology. After microangiography, all flaps were immediately immersed in 10% formaline for at least 3 days. Slices were cut perpendicularly to the long axis of the vascular pedicle at the proximal, middle and distal part of the flap, embedded in parafin and stained with haematoxylin eosin (HE), Elastica van Gerson (Evg) and Elastica Ladewig (Eldg). The morphological structures and all vessels perfused by micropaque were carefully observed and counted. Histological quantification was performed at 5! magnification using a 1 cm 2 eyepiece graticule under the light microscope.
Control group To determine the original vasculature in the abdominal skin flap, three rabbits (six flaps) were operated on in the control group. All the incisions were carried out in the same manner as in the study group, but there was no venous pedicle implantation. For microangiography, the control animals were heparinised intravenously and put under general anaesthesia. Plastic intravenous cannulas (0.5!0.9 mm2) were inserted into the carotid artery and jugular vein. Two thousand millilitres of heparinised saline heated to 38 8C was infused into the carotid artery at a standard pressure of 110 mmHg, then allowed to drain through the
In the study group, all abdominal flaps showed no specific findings during flap prefabrication. Necrosis and infections were not observed in any flap. Flap elevation as an island flap was demonstrated in two rabbits of the 20-day group. These elevated flaps showed no necrosis with inconspicuous wound healing and normal hair growth on the flaps similar to that seen in other nonoperated regions. A new fibrovascular connective tissue capsule underlying the flap could be identified in all flaps in the study group. With increased retention time of the pedicle, this fibrovascular capsule became thicker. As a result of this capsule, the arterialised venous pedicle and the abdominal fasciocutaneous flap became integrated, such that the implanted pedicles lay within the prefabricated flap.
Blood analysis A slight anaemia due to intraoperative blood loss was observed postoperatively in all animals of the study group as well as a decrease in haematological data such as WBC, RBC, HGB, HCT and MCV. There were no changes in the pre- and postoperative chemical data.
Selective microangiographic findings The results of vessel quantification from angiograms are presented in Table 1. In the 4-day group, a small number of newly formed vessels originating
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Table 1 Number of vessels counted from angiograms in the 4-, 8-, 12-, 16- and 20-day groups compared to the control group Group
Group Study group
4 days 8 days 12 days 16 days 20 days
Control group
A.N.o.V.
(%)
SD
A.N.o.V.
(%)
SD
2.6 84.5 107.9 175.13 252.34
1.7 35.5 44.1 73.28 105.58
G0.9 G11.8 G14.8 G19.1 G21.35
238.83
100
G5.82
A.N.o.V., average number of vessels; SD, standard deviation; %, percentage of counted vessels.
from the implanted pedicles could be identified (Fig. 2(B)). Eight days after pedicle implantation, the newly formed vessels were larger, more visible and some were connected to the originally available vasculature of the flap (Fig. 2(B)). In the 12-day group, newly formed vessels were more dense and exhibited wider-spread communication with the available vessel system. The vessel quantity in this group had increased to 44.81% compared to the control group (100%). By the 16-day group, newly formed vessels and their vascular connections had continuously progressed, resulting in a variety of different vessel sizes present within the flaps. The vessel quantity in this group reached a value of 73.28%. The 20-day group showed an even higher quantity and density in the vessel network with innumerable vascular communications and the vessel quantity increasing to 105.58% compared to the control group (Fig. 2(C) and (D)). At this point in the neovascular development, the entire prefabricated flaps were receiving blood flow supplied from the newly implanted venous pedicle.
Histological findings All the prefabricated flaps were vital and morphologically unconspicious with normal epidermis, hair follicles, connective tissue and blood vessels. In the 8-, 12-, 16- and 20-day groups, the flaps were slightly thickened due to the development of the new fibrovascular connective tissue capsules underlying the flaps (Fig. 3). This capsule developed from elastic collagen fibres with a rich capillary network. In the 8-day and 12-day group, the newly formed vessels had penetrated into the subdermal tissue of the flaps, especially around the implanted pedicle. In the 20-day group, the contrast medium spread out through newly formed vessels over the entire vascular system of the prefabricated abdominal flaps (Fig. 3). The
histological quantification was similar to that of the microangiographic findings.
Discussion Neovascularisation is a natural and indispensable part of wound healing. Although the precise mechanisms of neovascularisation are not well understood, it can be selectively combined with vascular implantation and microsurgical techniques to create customised neovascularised free flaps.15–17 Prefabricated free flaps have been investigated by different authors.18–27 Erol and Spira12 used omentum majus as sources of vascularity for skin grafts. In rat and rabbit models, Hirase et al.13 reported several types of neovascularised free flaps including muscle, bone, cutaneous, myocutaneous, fat and perichondrial flaps. Morrison et al.14 showed in an experimental study that neovascularisation began within 2 weeks and achieved maximum development by 8–12 weeks, irrespective of which vascular bundle, arteriovenous loop or artery was implanted below the elevated flap. Nichter et al.27 indicated from an experimental study on arterialised venous flaps in rabbits that arterial inflow is needed for predictable survival of composite tissue through the venous tree or the eventual ingrowth of recipient vessels into the flap by way of the delay phenomena. Based on the concept of a ‘flow-through’ vein, Takato et al.28 investigated the viability of prefabricated total venous perfusion flaps using the left epigastric vein in a rabbit model. They concluded that reproducible survival of prefabricated flaps can be obtained when the flap was elevated more than 3 weeks after prefabrication. Karatas and co-workers29 investigated the prefabrication of composite arteriovenous flaps with implantation of an autologous graft (cartilage) or an alloplastic material (porous
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637 polyethylene) in rabbits, and showed that arteriovenous perfusion can nourish a prefabricated flap containing an implanted material (autologous or alloplastic) and that 2-week delayed composite flaps have a similar survival rate to delayed prefabricated conventional axial flaps. Applying a different model as these investigators, we have created prefabricated flaps in Chinchilla Bastard rabbits as large as 8!15 cm2 using an isolated vein dissected from the femoral and saphena magna vein, arterialised by end-to-end anastomosis to the femoral artery at the inguinal ligament, as an implanted vascular pedicle. A silicone sheet with the corresponding dimension of 8 cm!15 cm!0.25 mm was placed on the abdominal wall to prevent angiogenesis from occurring between the underlying vascular bed and the abdominal flap.30 The selective microangiographic and histological results showed that newly formed vessels arising from the implanted pedicles were already evident 4 days after pedicle implantation and silicone insertion. With increased retention time of the pedicle, they began communicating with the originally available vasculature in the abdominal prefabricated flap to establish a newly neovascularised-rich system. Twenty days after implantation, the results of macroscopic observation, microangiographic and histological findings showed that the entire abdominal fasciocutaneous flap was well perfused by the blood flow supplied from the newly implanted arterialised venous pedicle through newly formed vessels and their vascular connections. At this point in neovascular development, the vessel quantity on microangiograms had increased to 105.58% compared to the control group. The newly neovascularised system in the flaps consisted of the implanted pedicle, newly formed vessels, the originally available vasculature and their rich vascular connections. Our
Figure 2 (A) Selective microangiography of the 4-day group. (B) Selective microangiography of the 8-day group. Newly formed vessels arising from the implanted pedicle were connected with the originally vasculature in the abdominal fasciocutaneous flap. (C) Selective microangiography of the 20-day group showed the newly neovascularised system in the prefabricated flap. The entire vascular system of the flap was perfused by the blood flow supplied from the newly implanted arterialised venous pedicle. (D) Microangiography of the control group shows the originally available vasculature in the abdominal fasciocutaneous flap.
Figure 3 Histologic specimen of the 20-day group (EvG—stain). The contrast medium spread out through newly formed vessels into the entire vasculature of the flap.
638 observations were similar to those of Itoh,31 who showed in an experimental model in rats, that newly formed vessels were generated not only from the implanted vascular pedicle, but also from the original vessels of the flap. Most of the vascular proliferation occurred within the first week. An important question related to the clinical application of prefabricated flaps using isolated arterialised venous pedicles, is how long it takes for the matured neovacularisation process to occur in the flap after pedicle implantation. In the present study, selective microangiography demonstrated that 20 days after pedicle implantation, the entire flap was perfused by the blood flow supplied from the implanted pedicles. Histological evaluations of the 20-day group showed a normal skin subdermal architecture with the micropaque spreading out over the entire vascular system in the flaps. Our results indicate that 20 days is the minimal length of time required after pedicle implantation for maturation of neovascularisation in prefabricated flaps using silicone sheets and isolated arterialised venous pedicles. The influence of the panniculus carnosus on the increase in neovascularsation in prefabricated flaps was reported by many authors.4,16,18,31 The paniculus carnosus, with its rich capillary vascular network, can play an important role in establishing vascular connections in the flaps. For clinical applications, the fascia underlying the flap must be protected to stimulate the neovascularisation processes in these flaps. The technique of flap prefabrication using isolated arterialised venous pedicles for clinical applications in defect reconstruction is quite promising. This procedure allows surgeons to select implanted vascular pedicles very easily and safely based on the abundant venous system of the human body. Applying the principles of the prefabricated flap, custom-made neovascularised flaps using isolated arterialised venous pedicles can be created in desired regions of similar colour, thickness and dimension regardless of its native vascular anatomy. The technique results in a reduction in functional morbidity and more aesthetic donor defects. Disadvantages of the method are the need for a two-stage operation and the scar in the venous pedicle donor site. Flap prefabrication by means of pedicle implantation into a random-pattern vascularised tissue area represents a new and powerful tool for reconstructive surgeons. The technique allows the creation of reliable axial well-revascularised flaps, which satisfy specific requirements for defect reconstruction. Based on this study, it can be concluded that an isolated arterialised venous
N.T. Hoang et al. pedicle can be used as an implanted vascular pedicle in a prefabricated flap. Twenty days is the minimal period of time required after pedicle implantation for the maturation of the neovascularisation in prefabricated flaps using silicone sheets and isolated arterialised venous pedicles. These experimental results have shown the promising potential of isolated venous pedicles for clinical applications.
Acknowledgements This work was financially supported by the DFG (Deutsche Forschungsgemeischaft). We also wish to thank Dr K. Herfeldt and Dr O. Petrowicz—Institute for Experimental Surgery of the University Hospital rechts der Isar of the Technical University of Munich, Germany for their help and advice in performing the histological images and statistical analysis.
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Prefabrication of large fasciocutaneous flaps 12. Erol O, Spira M. Omentum island skin graft flap. Surgical Forum 1978;29:594–6. 13. Hirase Y, Valauri FA, Buncke HJ, Newlin LJ. Customized prefabricated neovascularized free flaps. Microsurgery 1987;8:218–24. 14. Morrison WA, Dvir E, Doi K, Hurley MJ, O’Brien B. Prefabrication of thin transferable axial-pattern skin flaps: an experimental study in rabbits. Br J Plast Surg 1990;43: 645–54. 15. Atabey A, Mc Carthy E, Manson P, Kolk CAV. Prefabrication of combined composite (chimeric) flap in rats. Ann Plast Surg 2000;45:484–90. 16. Tanaka Y, Sung KC, Tsutsumi A, Ohba S, Ueda K, Morrison WA. Tissue engineering skin flaps: which vascular carrier, arteriovenous shunt loop or arteriovenous bundle, has more petential for angiogenesis and tissue generation? Plast Reconstr Surg 2003;112:1626–44. 17. Nguyen T. Hoang. Die Neovaskularisation im praeformierten Gewebelappen in Abhaengigkeit von arteriovenoesen Blutfluss des implantierten Gefaesstieles. Dissertation TU Munich; 1997, p. 1–127. 18. Shen Z, Wang S, Cheng X, Lu J, Yin D, Sun Y. Greater omentum-cutaneous axial flap: a method to create transferable skin flap. Chin Med J 1981;94:718–22. 19. Erk Y, Rose FA, Spira M. Vascular augmentation of skin and musculotaneous flaps. Ann Plast Surg 1983;10:341–8. 20. Kwan CT, Khouri RK, Keuk SS, Shaw WW. The fasciovascular pedicle for revascularisation of other tissues. Ann Plast Surg 1991;26:149–55. 21. Maitz PKM, Pribaz JJ, Duffy FJ, Hergrueter CA. The value of the delay phenomenon in flap prefabrication: an experimental study in rabbits. Br J Plast Surg 1994;47:149–54.
639 22. Adams WP, Griffin JR, Friedman RM, Rohricht RJ, Robinson JB. The myoadipose flap: a new composite. Plast Reconstr Surg 1998;102:735–40. 23. Hickey MJ, Wilson J, Hurley JV, Morrisson WA. Mode of vascularisation of control and basic fibroblast growth factorstimulated prefabricated skin flaps. Plast Reconstr Surg 1998;101:1296–304. 24. Alm MI, Asahina I, Seto I, Oda M, Enotomo S. Prefabricated vascularized bone flap: a tissue transformation technique for bone reconstruction. Plast Reconstr Surg 2001;108:952–8. 25. Hong JP, Lee HB, Chung YK, Kim SW, Tark KC. Coverage of difficult wounds around the knee joint with prefabricated, distally based sartorious muscle flaps. Ann Plast Surg 2003; 50:484–90. 26. Macleod TM, Williams G, Sanders R, Green CJ. Prefabricated skin flaps in rat model based on dermal replacement matrix Permacole. Br J Plast Surg 2003;56:775–83. 27. Nichter LS, Haines PC. Arterialized venous perfusion of composite tissue. Am J Surg 1985;150:191–6. 28. Takato T, Komuro Y, Yonehara H, Zucker RM. Prefabricated venous flap: an experimental study in rabbits. Br J Plast Surg 1993;46:122–6. ¨ , Atabey A, Demirdo 29. Karatas O ¨ver C, Barutcu A. Delayed prefabricated arterial composite venous flaps: an experimental study in rabbits. Ann Plast Surg 2000;44:44–52. 30. Braley SA. The use of silicones in plastic surgery. Plast Reconst Surg 1973;51:280–8. 31. Itoh Y. An experimental study of prefabricated flaps using silicone sheets, with reference to the vascular patternization process. Ann Plast Surg 1992;28:140–6.