New experimantal composite flap model in rats: Gluteus maximus-tensor fascia lata osteomuscle flap
NEW EXPERIMANTAL COMPOSITE FLAP MODEL IN RATS:GLUTEUS MAXIMUS-TENSOR FASCIA LATA OSTEOMUSCLE FLAPSERDAR NASIR, M.D.,1* ASIM AYDIN, M.D.,1AYCAN KAYIKCIOGLU, M.D.,2 CENK SOKMENSUER, M.D.,3 ANDAZEM COBANER, M.D.4Experimental animal models need to be developed forstudies of composite flaps that have often recently been usedfor defects of both bone and soft tissues. A consistentanatomy, simple surgical technique, and reliable blood floware essential for the success of experimental flap studies.Here we propose a gluteus maximus-tensor fascia lata os-teomuscle flap in rats as a model of these qualities. Gluteusmaximus and tensor fascia lata muscles and the adjacentiliac bone segment were combined as a lateral circumflexfemoral artery-based flap. To test the reliability of this com-posite flap, three types of composite tissues were harvestedand replaced: osteomusculocutaneous flap, osteomuscleflap, and osteomuscle composite graft. The osteomusculo-cutaneous flap was elevated by including a skin island overthe gluteal region. The osteomuscle graft was formed bydeliberately dividing the vascular pedicle of the osteomuscleflap. Direct observation revealed complete necrosis ofthe skin islands in all osteomusculocutaneous flaps.Microangiography of the flap demonstrated that both mus-cles and the attached bone were supplied by the pedicle. Dyestudies with nitro blue tetrazolium (NBT) and India ink dem-onstrated dye uptake2 in both muscle and bone componentsin osteomuscle flaps. Histological examinations also dem-onstrated the viability of both tissues only in the flap group.Bone scintigraphy performed in flaps on postoperative day 7demonstrated radionuclide uptake, confirming perfusion ofthe bony segment. The gluteus maximus-tensor fascia lataosteomuscle flap is a reliable and simple model for com-posite flap studies that offers the following advantages: 1) itis a new composite flap which includes bone, 2) it can bedissected easily with the naked eye, without using themicroscope, 3) it has a long pedicle for flap displacement,and 4) it is a small animal model. 2003 Wiley-Liss, Inc.MICROSURGERY 23:582588 2003Osteomuscle flaps have been used in the reconstructionof complex defects after either tumor extirpation or se-vere trauma.17 Clinical applications of osteomucleflaps have fueled an interest in the biology of these flaps,particularly the mechanisms by which the bone cellsurvives and heals bone defects with a resistance to ra-diotherapy.8 Further investigations of osteogenic, oste-oinductive, and osteoconductive substances are alsobeing conducted. For this reason, experimental flap re-search is still needed for the proper use of compositeflaps in reconstructive surgery.911 Therefore, some ex-perimental flap models were described in small ani-mals.1217Rats are preferred for their affordability, easy han-dling, and robustness. On the other hand, the maindisadvantage of small animal flap studies is the re-quirement of tedious dissection under a microscope.Extended surgery and anesthesia time by using a mi-croscope in studies with large numbers of animalsmay become frustrating. Increased working time mightcause the animals to die. An easily and quickly dis-sected flap, by obviating the need for surgical expertise,would let researchers focus on the main theme of theirstudy.Although many muscle and myocutaneous flapmodels were described,19,20 only four osteomusculocu-taneous flap models were reported in the rat: the saph-enous artery flap,12 the iliac osteomusculocutaneousflap,13 the thigh flap,14 and the acromiotrapezius flap.15The purpose of this study was to describe an osteo-musculocutaneous flap model in rats which can be dis-sected quickly with the naked eye for smoothercomposite flap studies.1Department of Plastic and Reconstructive Surgery, Suleyman Demirel Uni-versity School of Medicine, Isparta, Turkey2Department of Plastic and Reconstructive Surgery, Hacettepe UniversitySchool of Medicine, Ankara, Turkey3Department of Pathology, Hacettepe University School of Medicine, Ankara,Turkey4Nuclear Medicine, Antalya, Turkey*Correspondence to: Dr. Serdar Nasr, _Istiklal mahallesi 105. cad. No: 62Daire:1 4 Isparta, Turkey. E-mail: email@example.comReceived 26 May 2003; Accepted 8 July 2003Published online in12 Wiley InterScience (www.interscience.wiley.com). DOI:10.1002/micr.10212 2003 Wiley-Liss, Inc.MATERIALS AND METHODSAnimal CareIn total, 40 adult Wistar rats weighing 200250 gwere used in this study. This study was conductedaccording to the guidelines of the University AnimalCare Committee. All animals were kept in individualcages in a room with standard environmental condi-tions. The animals were fed ad libidum. Operations wereperformed under ketamine (90 mg/kg) and xylazine (10mg/kg) anesthesia. Supplementary doses were given asnecessary.SurgeryRats were divided into three groups: anatomic study,flap group, and graft group.Anatomic Studies (n= 10)Animals were dissected for vascular anatomy of thegluteus maximus3 and tensor fascia lata (TFL) muscles,and the branching pattern of their pedicle and lateralfemoral circumflex artery. Dissection of the vascularpedicle was carried from distal to proximal, until theorigins of the vessels from the abdominal aorta, andinferior vena cava were identified. A laparatomywas performed to measure the diameter and the lengthof the vascular pedicle. Dimension and weight of thegluteus maximus tensor fascia lata muscles were alsomeasured.Flap Group (n= 20)Two types of flaps were designed: an osteomuscu-locutaneous flap (n = 10) and a osteomuscle flap (n =10). For the former type, a skin island was planned,measuring about 3 3 cm over the gluteus maximusmuscle, extending from the dorsal midline to the ante-rior axillary line, and from the hip joint to the superiorventral spine of the iliac bone (Fig. 1A). Following in-cision of the skin island and stitching it to the underlyinggluteus maximus muscle to avoid stretching of skinperforators, the gluteus maximus muscle was dividedfrom its insertion to the hip joint. Then the muscle wasdissected anteriorly, including the inseparably unitedtensor fascia lata muscle. The fascia in the mid-dorsalside of the rat was incised parallel to the vertebrae up tothe superior ventral spine of the iliac bone. Then thegluteus maximus muscle was divided from the thighfascia anteriorly and the biceps femoris muscle posteri-orly. The Iliac bone osteotomy was planned withoutdisrupting the muscle-bone connection. A portion ofiliac crest measuring 1 1 cm was included in the flap.Consequently the flap was elevated over the lateralfemoral circumflex artery that enters the flap betweenthe rectus femoris and iliacus. All components of theosteomusculocutaneous flap, including the gluteusmaximus muscle, tensor fascia lata muscle, iliac bonesegment with its muscle cuff, and the overlying skin is-land, were elevated on the lateral femoral circumflexvascular vessels as a single unit (Fig. 1BD). The othertype of flap was the osteomuscle flap comprising onlymuscle and bone without including a skin island. Theisland flaps were replaced.Composite Graft Group (n= 10)The osteomuscle flap as previously described waselevated, and then the vascular pedicle was divided inthe zone between the rectus femoris and iliacus muscle.Thus, muscle and iliac bone segment were replaced.Assessment of Flap ViabilityDirect observation. The survival of the skin islandwas evaluated on postoperative day 7 by gross inspec-tion of the flap color compared with normal skin.21Microangiography. Microangiography was applied todelineate the vascularization of the osteomuscle flap intwo animals on postoperative day 7. After cannulatingthe abdominal aorta, 50 cc of the mixture (at 70C) thatwas made of 50 g of silver oxide, 5 g of gelatin, and 100 ccof the isotonic was injected with a syringe. The flaps werestored in an animal refrigerator at 4C for 4 h. Then allflaps underwent radiography with an animal soft X-raymachine, using Microvision-C mammography film.Dye studies. Nitroblue tetrazolium staining was usedfor muscle viability on postoperative day 7. Accord-ingly, muscle tissues from three animals of each groupwere sliced into 0.5-cm pieces and were held for 30 minat 37C in buffered nitroblue tetrazolium. Ischemic in-jury between flap and graft groups was compared ac-cording to stain quality with nitro blue tetrazolium5(NBT).Viability of bone was evaluated by the presence ofdye in the vessels after India ink injection. Followingcannulation of the abdominal aorta in three animalsfrom the ostemuscle flap group with a 24-gauge cathe-ter, 5 ml of India ink were injected. The bone segment ofthe flap was divided, decalcified, stained with hema-toxylin-eosin, and examined under light microscopy forNew Experimental Composite Flap Model in Rats 583the presence of India ink in blood vessels of the bonemarrow and cortex.Bone scintigraphy. In the flap group, three animalsunderwent radionuclide bone scintigraphy on post-operative day 7. After the animals were anesthetized,1 mCi6 of techenitium-99m methilene diphosphonate wasinjected via the external jugular vein. Approximately 4 hafter the injection, scanning was carried out using agamma camera with a low-energy, general-purposeparallel-hole collimator.17Histology. On postoperative day 7, the animals in theflap and graft groups were killed with an overdose ofinhaled ether. Flaps and grafts were harvested andplaced in 10% buffered formalin. Bone segments weredecalcified in a mixture of formalin, formic acid, andconcentrated hydrochloric acid. The bone and musclesections stained with H&E were evaluated.RESULTSAnatomic StudiesThe gluteus maximus muscle is a thin muscle arisingby a fascia from the dorsal border of the ileum, from thelast three sacral and the first caudal vertebrae. Ante-riorly, it becomes inseparably united with the tensorfascia lata; posteriorly its origin is covered by the ante-rior head of the biceps femoris. It inserts by a tendon tothe greater trochanter of the femur. Superiorly, it isFigure 1. A: Skin island designed over gluteus maximus muscle. B:Gluteus maximus-tensor fascia lata osteomuscle flap harvested onlateral femoral circumflex artery. C: Gluteus maximus-tensor fascialata osteomuscle flap isolated on lateral femoral circumflex artery. D:Schematic presentation of11 flap. SVS, superior ventral spine of iliacbone; HJ, hip joint; S, skin; B, bone; M, muscle; P, pedicle.584 Nasir et al.connected to the iliac bone by means of the superiorventral spine.22 The muscle is of triangular shape, withan average size of 32 29 27 mm and weighing, onaverage, 1.12 g. In preliminary anatomic dissections, abone segment which included the superior ventral spineof the iliac bone was resected with an average 10 mmlength, 10 mm height, and 1 mm thickness by preservingthe connection between the muscle and bone (Fig. 2).The lateral femoral circumflex artery is the vascularpedicle of the gluteus maximus muscle in rats, andsprings from the hypogastric artery instead of from thefemoral as in humans. The hypogastric artery, giveslateral femoral circumflex artery branch after the inter-nal iliac-external iliac bifurcation. The lateral femoralcircumflex artery leaves the pelvic cavity, passing underthe psoas major muscle and dividing into two branches.One of the branches springs into twigs in surroundingmuscles. The other branch becomes superficial on themedial aspect of the thigh, appearing from between theiliacus and rectus femoris, and ends as a terminal branchin the gluteus maximus. The veins accompany the arteryand open into the hypogastric vein. The diameters of thelateral femoral circumflex artery and vein at their originswere 0.10.2 mm and 0.20.4 mm, respectively. Theaverage length of the vascular pedicles from the origin tothe hilus of the muscle was found to be 3.0 cm.Flap and Graft StudiesDirect observation on postoperative day 7 of theskin islands of all osteomusculocutaneous flaps wascompletely necrotic (Fig. 3).Microangiography revealed the vascular supply inboth bone and muscle components as provided by thepedicle (Fig. 4).Muscle staining with nitroblue tetrazolium revealedthat the muscles of the flap group were dark blue due toink uptake, although those of the graft group were notdyed by NBT, indicating the viability of the musclecomponent in the flap groups as opposed to graft group(Fig. 5).Dye study with India ink at postoperative day 7demonstrated ink uptake by the vessels in the bonemarrow and cortex of the bone, confirming bone per-fusion through the connection between muscle and bone(Fig. 6).Figure 2. Anatomy of gluteus maximus muscle in rat. GM, gluteusmaximus; TFL, tensor fascia lata; BF, biceps femoris; SVS, superiorventral spine of iliac bone; ST, semitendinosus.Figure 3. Necrotic skin component of flap (arrows) 7 days afteroperation.Figure 4. Microangiographic analysis reveals course of pedicle. M,muscle; B, bone; P, vascular pedicle.New Experimental Composite Flap Model in Rats 585Bone scintgraphy demonstrated radionuclide uptakeby the bone segment, indicating the viability of the bonycomponent of the flaps (Fig. 7).Histological examination of the muscle and bonesegment on postoperative day 7 showed normal-appearing muscle cells and viable osteocytes within thelacunae and bony trabeculae. However, examinationunder a light microscope of the graft group demon-strated nonviable, necrotic muscle cells and empty la-cunae with no visible osteocytes (Fig. 8A,B).The summary of results for these postoperativeperfusion and survival tests is given in Table 1.DISCUSSIONComposite flaps containing vascularized bone havesome advantages over nonvascularized bone grafts.Higher survival rates of the cellular elements, minimalbone resorption, and faster and better callus formationwere demonstrated in the vascularized bone grafts.21 Infree bone graft transfers, most of the bone cells exceptthose immediately subjacent to the periosteum perished,and the dead bone acted as a scaffolding for the in-growth of new cells. Because of the poorly vascularizedFigure 5. Muscle staining with NBT. Muscles of flap were staineddark blue (bottom row); muscles of graft were not stained (upperrow).Figure 6. Histological examination of bone marrow and cortex fromflap group indicates perfusion of bony component, evidenced bypresence of ink in vessels (arrow).Figure 7. Radionuclide bone scan demonstrates radioisotope up-take of bony component of flap bilaterally (arrows).Figure 8. A: Histological section. Musculo-osseous part of flap onpostoperative day 7 demonstrates viability of each segment. B:Histological examination of graft group on postoperative day 7.Nonviable, necrotic muscle cells and empty lacunae in bone areobserved. Muscle cell, white arrow; osteocyte, black arrow.586 Nasir et al.tissue bed often found in the reconstruction of com-posite defects, reconstruction with a flap consisting ofbone gives more pleasing results than with a soft-tissueflap over a bone graft.23,24 These findings promote morefrequent use of composite flaps. Despite their commonuse (and remarkable advances have been made invascularized bone flap transfers), many basic sciencequestions need to be resolved. Therefore, developmentof a proper experimental animal model for basic scienceresearch is a critical step.Although composite flaps which include a bonesegment, such as osteomuscular, osteomusculocutane-ous, and osteocutaneous,25 were described in recon-structive surgery, only four osteomusculocutaneous flapanimal models were reported in the rat: the saphenousartery flap, iliac osteomusculocutaneous flap, thigh flap,and acromiotrapezius flap.1215 All flaps are appropri-ate for scientific studies related to the biology of bonecells. However, flap dissections are made using a mi-croscope due to flaps complicated surgical anatomies.This situation is disadvantageous for osteomuscular flapstudies, which use wide animal series.The gluteus maximus muscle is the most superficialmuscle in the gluteal region of the rat. The muscle isbounded superiorly by the superior ventral spine of theiliac bone, inferiorly by the greater trochanter of thefemur, posteriorly by the fascia from the dorsal borderof the ileum, and anteriorly by the thigh fascia. Theentrance of the vascular pedicle is approximately themiddle region of the muscle. Because of the consistentlandmarks and clearly defined vascular pedicle, thegluteus maximus muscle is elevated easily as an osteo-muscle flap without using a microscope. In our labora-tory, the complete surgery of this flap was performed inabout 10 min. Using an experimental flap which can bedissected quickly with the naked eye in rats would makea great contribution to composite flap studies.The long pedicle allows a greater range of flapplacement from the superior ventral spine of the iliacbone to the knee. Besides, a muscle mass of 1.12 g inaverage sufficiently supports studies requiring bio-chemical assays. The associated8 iliac bone segment of 10mm length, 10 mm height, and 1 mm thickness is suit-able for bone studies.Battal et al.26 raised the gluteus maximus muscle as amusculocutaneous flap with a skin island. They claimedcomplete survival of skin islands in their study onpostoperative day 10. Although skin islands were ele-vated as described,26 we observed total skin necrosis inall flaps at postoperative day 7 (n = 10 rats). Moreover,no musculocutaneous perforators between the gluteusmaximus and overlying skin were determined under asurgical microscope. This condition might show that glu-teus maximus osteomuscle flaps are not true osteomus-culocutaneous flaps. On the other hand, we did not findthis flap suitable for free transfer, contrary to the sugges-tion by Battal et al.26 for the very small diameter andmul-tiple branches of the lateral femoral circumflex artery.The gluteus maximus-tensor fascia lata osteomuscleflap of the rat offers the following advantages:1. The flap is harvested from a small animal species.2. The flap can be elevated quickly, with the naked eye.3. The flap, which includes a bone segment, is a newcomposite flap and suitable for bone studies and bi-ochemical assays.4. The long pedicle allows a greater range of flap dis-placement.This flap also has some disadvantages: it is notsuitable for free transfer, and no skin island can beincluded.As an osteomuscle unit, we demonstrated the via-bility of muscle and bone segment by NBT, dye injec-tion, histological examination, radionuclide bone scan,and microangiography. The reliability of these methodsin demonstrating the perfusion and viability of bone andmuscle was confirmed in this study.We recommend the gluteus maximus-tensor fascialata osteomuscle flap as a reliable and simple model forcomposite flap studies in rats.Table 1. Postoperative Test Results for Tissue Perfusion and Viability*Postoperative testsGroup Skin observationMuscle staining(NBT)Dye study(India ink) MicroangiograpyBonescintigraphyHistologicalexaminationOMC flap Total skin necrosis Dark blue Visualizedin vasculatureVisualized flapvasculatureRadionuclide uptake Viable muscle cellsand osteocytesOM flap Not applied Dark blue Visualizedin vasculatureVisualized flapvasculatureRadionuclide uptake Viable muscle cellsand osteocytesOM graft Not applied Not stained Not visualizedin vasculatureNot applied Not applied No viable musclecells and osteocytes*OMC osteomusculocutaneous; OM, osteomuscle.New Experimental Composite Flap Model in Rats 587REFERENCES1. Baker SR. Reconstruction of mandibular defects with the revas-cularised free tensor fascia Iata osteomyocutaneous flap. ArchOtolaryngol 1981;107:414418.2. Urken ML, Weinberg H, Vickery C, Buchbinder D, Lawson W,Biller HF. 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J Reconstr Microsurg 1997;13:251255.588 Nasir et al.