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Int. ,L Oral Maxillofac. Surg. 1999; 28:469~174 Printed in Denmark.

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Copyright 9 Munksgaard 1999 [ntemadonalJoumalof

Oral & Maxiflofacial Surgery
ISSN 0901-5027

Microvascular network of the healing surface over the temporalis flap maxillary reconstruction
L. K. Cheung: Microvascular network o f the healing surface over the temporalis flap in maxillary reconstruction. Int. J. Oral Maxillofac. Surg. 1999; 28:469 474.

Lim-Kwong Cheung
Oral & Maxillofacial Surgery, Faculty of Dentistry, University of Hong Kong, Hong Kong SAR, China

9 Munksgaard, 1999
Abstract. This study aims to describe and quantify the microvascular changes of

the healing wound i n / l maxilla reconstructed by a temporalis myofascial flap (TMF). 24 eats underwent partial maxiliectomies and reconstruction by T M E Vascular infusion was performed immediately following sacrifice and then analyzed by radiography, light and SEM. ReSults showed that the superficial microvascular layer overlying the T M F underwent a similar transformation as in the histological healing sequence. The vascular density was highest during the initial 6 weeks, followed by reducing density at the chronic inflammatory phase, to normal at the proliferative and remodelling phases. The migrating epithelium brought on a mi~rovascular sub-epithelial plexus, which eventually covered the TMF. The finding that the vascular source of this sub-epithelial plexus comes from the surrounding tissues rather than from the underlying T M F will have surgical implications for bony reconstruction of the maxilla.

Key words: temporal muscle; surgical flaps; microvascular.

Accepted for publication 15 July 1999

Survival of a pedicled tissue flap depends on the integrity and adequacy of its blood supply following the transfer process. Temporalis muscle has been shown to have a rich intramuscular microvasculature supplied by 3 primary arteries, namely the anterior deep temporal, posterior deep temporal and middle temporal arteries 3. The two former arteries are branches derived from the internal maxillary artery, whereas the last one is derived from the superficial temporal artery ~,2. The temporalis myofascial flap (TMF) has been proven to be very reliable clinically for reconstruction of oral defects with minimal vascular complications during the healing process 7,8,1~ An animal model of T M F in cats has previously been developed and the heal-

ing process simulates the human situation 5, The source ofmucosa healed over the T M F has been confirmed as deriving frona the epithelial cells from the surrounding wound margins 6. However, the origin of the microvascular plexus underneath this repaired oral mucosa remains unknown. Considerable vascular change is expected to occur in the T M F during the different phases of the histological healing process, but this aspect still remains to be defined. The aims of this study were to describe the microvascular changes of the healing wound in the maxilla reconstructed by the TMF, and to assess the differences between the vascular network underneath the repaired mucosa of the T M F and that below the normal palatal mucosa.

Material and methods A total of 24 cats of the Felis catus species were used for this study. They all underwent standardized partial maxillectomies and reconstruction with the ipsilateral TME The surgical technique of this operation in cats has been reported previously5. All flaps healed progressively without clinical complications of infection, wound dehiscence or fistula formation. The cats were killed at defined post-surgical intervals in weeks 2, 4, 6, 8, 12, 14, 18 and 24. Each cat was killed by an overdose of ketamine at the determined time. Both sides of the neck were surgically explored and the external carotid arteries were cannulated with gauge 18 intravenous catheters and secured by multiple ligatures. The blood in the vascular system of the head and neck was flushed out by a slow infusion of heparinized saline solution. This was followed by injection of one of three

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thickness of the specimens and the images were captmzd using instant films (Polaroid 550 positive & negative films, USA). The Indian ink infused specimens were embedded in paraffin blocks and prepared into thin sections of 10/~m thickness and thick sections of 300 #m thickness. The thin sections were stained with haematoxylin and eosin (H&E) and the thick sections remained unstained; both were mounted on glass slides. The speci-

infusion media: Indian ink solution, lead oxide solution or methyhnethacrylate resin. The composition of the infusion media and the methods of preparation of the maxillary specimens have been reported previously4. The cats were randomly assigned into one of 3 groups and the choice of infusion medium was made according to the defined sacrifice time for that group. The lead oxide solution was selected to illustrate the arterial pattern in the TMF following its transfer into the oral cavity for maxillary reconstruction. The Indian ink solution would demonstrate the microvascular network and the changes of the TMF during the healing process. The methylmethacrylate resin would form vascular corrosion casts to complement the result of the Indian ink findings and to illustrate the microvasculatures in three dimensions underneath the repaired oral mucosa and compared with the contralateral normal palatal mucosa. The lead oxide infused head specimens were radiographed (Hewlett Packard x-ray system 43805N, USA) at 70 kvp for 2-5 minutes. The exposure time was adjusted according to tire

Fig. 1. Macrovascular network changes following temporalis flap transposition to maxilia by lead oxide infusion. On the radiographic submental vertex view following removal of the mandible in cat, the right side (R) was the normal and the left (L) was the operated side. On the normal side, the path of the internal maxillary artery is illustrated with its branches supplying the maxilla: (a) internal maxillary artery, (b) infra-orbital artery, (c) posterior palatine artery, (d) descending palatine artery, and (e) sphenopalatine artery. However, on the operated side, obvious medial displacement of the internal maxillary artery by the muscle was noted. Fine branches derived directly from the rete mirabile (*) and the internal maxillary artery were seen.

Fig. 2. Microvascular changes in the superficial layer of the temporatis flap during the healing process. A: In the acute inflammatory phase during the first 6 weeks, the microvasculatures of the superficial layer were dilated, forming bush-like bundles. The vascular contribution to this layer was derived from the muscle flap rather than the normal side of the palate (Indian ink x20). B: Histological section of the corresponding palate/flap junction as in A. (H&E • 50). C: In the chronic inflammatory phase during the 8th-14th weeks, the dilated microvascular bundles above the temporalis muscle fibres became more discrete but still separated from the surface by an avascular layer. New microvasculatures (I~) were introduced by the migrating epithelium from the palatal side (Indian ink • D: Histological section of similar site as in C, showing onset of chronic inflammatory phase on the flap (H&E • E: At higher magnification of the advancing front at the buecal side of the flap, details of the new vascular plexus (*) could be seen (Indian ink • F: Histological section of similar site as in E, showing epithelial migration and hyperplastic changes (H&E ?<50). G: During the proliferative phase on the flap centre, the vasculatures of the superficial vascular layer reduced in density (Indian ink • H: Histological section of the central region of the flap similar to that in G (H&E x20).

Microvasculatures of healing T M F
mens were examined under light microscopy (Leitz Orthoplan, Germany). The corrosion vascular casts infused by methylmethacrylate resin were sectioned into small seganents and mounted on copper stubs with conductive colloidal carbon. The casts were sputter coated with gold and examined under scanning electron microscopy (Joel JXA-840 SEM, Japan). In addition to the morphological description of the microvascular networks at the different healing times, morphometric analysis of the vascular density in the Indian ink infused thick sections was performed. The images of the vascular networks were captured through a video camera (JVC TK-1280E, Tokyo, Japan) mounted on the microscope (Nikon Ophtihot, Japan) to a computerized image analyzer system (Leica Quantimet 500+, Cambridge, England). A standard field was created and the microvascular networks within this field were quantified as the vascular density percentage. The vascular density at the wound margin, wound centre and contralateral normal palatal mucosa was measured 5 times from separate slides at each of the sacrifice times. The means and standard deviations of the vascular density percentage of the three selected fields were compared by one-way analysis of variance (ANOVA). If the P value was less than 0.05, then further comparison of the groups was conducted by a Tukey-Kramer multiple comparison test to identify the significant pairs. Instat software (Instat Ver 3.0, GraphPad Software Ltd., San Diego, USA) was used for the statistical analysis.

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Results With the T M F transposed into the oral cavity, the most obvious change was the medial displacement of the internal maxillary artery by the muscle bulk (Fig. 1). The n o r m a l arrangement of the anterior palatine artery and its branches disappeared and was replaced by fine branches derived from the rete mirabile or directly from the internal maxillary artery further back. This indicates the maintenance of macrovascular supply to the T M F from its original source with no significant alteration during the healing phases. The muscle fibres remained well perfused by microvessels as confirmed by the Indian ink infusion of the fibres. The healing T M F was shielded from the oral cavity by a thick layer of vascular tissue at all times during the healing process. This superficial vascular layer was found to change its pattern and density, correlating with the different phases of histological healing. In the acute inflammatory phase of the T M F healing during the first 6

Fig. 3. Comparison of sub-epithelial microvascular plexus between the repaired mucosa and normal palatal mucosa at 6 months postoperatively. A: The temporalis muscle remained wellperfused and was covered by a flat mucosa (Indian ink ?420). B: The microvascular network on the normal side of hard palate (Indian ink ?<20). C: The microvascular plexus beneath the repaired mucosa was separated from the underlying muscle fibres by a less vascular layer. Contribution to this plexus was derived from the buccal and palatal sides of the maxilla (Indian ink • D: The microvascular network in the lamina propria of the normal palatal mucosa was derived from the submucosal vascular plexus underneath (Indian ink x50). E: With the removal of the mucosal tissues by a corrosion process, the aerial view of the subepithelial microvascular plexus was observed as a fine interlacing network of capillaries connected by branching arterioles and venules (SEM • F: At the normal side of the hard palate, transverse vascular ridges corresponding to the rugae were present (SEM ?<20). G: With higher magnification of the microvascular plexus beneath the repaired mucosa, mild undulation of the capillaries could be observed at an oblique angle (SEM • H: The capillaries formed tall hairpin loops at the normal side (SEM • 150).

weeks, the microvascular diameters of vessels of the superficial vascular layer dilated enormously and appeared bushlike with anastomosing loops at the top of each bundle (Fig. 2a, b). The micro-

vessels were separated from the surface by a thin layer of avascular tissue. The vascular source of the superficial microvascular network was derived from the T M F below and the buccal tissue lat-

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connected by branching arterioles and venules (Fig. 3e). A layer of larger vessels could be seen below this superficial microvascular network and they corresponded with the intramuscular vessels of the T M E At an oblique angle view of the superficial vessels, they demonstrated short hairpin folding of the capillaries (Fig. 3g). In contrast, the normal hard palate had undulating ridges of capillaries grouping together, which corresponded to the palatal rugae (Fig. 3f). On the surface of this vascular layer, the capillaries formed tall loops of hairpin configuration (Fig. 3h). The vascular density of the superficial microvascular layer at different time periods of T M F healing is illustrated in Fig. 4. The vessels at the flap margin and the flap centre were statistically significantly different from the normal side (P<0.001) during the first 8 weeks, after which there were no obvious differences between the groups. When the vascular density was compared between the flap margin and the flap centre during the same period, there were no significant differences throughout the healing process except at the 6th week (P<0.05). When the flap margin vascular density was assessed on its own, there were no statistically significant changes during the first 6 weeks. The vascular density started to reduce from the 8 th week, stabilized at the 14th week and remained the same until the 24 th week. Similar findings were noted in the vascular density at the flap centre over time.
Discussion

erally, with no apparent contribution from the palatal side. With the progressive change into the chronic inflammatory phase during the 8th-14th weeks, the dilated vascular bundles above the temporalis muscle fibres became more discrete but were still separated from the surface by an avascular layer. Formation of a new vascular plexus was noted below the advancing epithelium at the palatal and buccal sides of the flap (Fig. 2c-f). At the 18 th week, the vessels in the superficial vascular layer had become less dense and smaller in diameter, while the flap was progressing through the proliferative phase of healing (Fig. 2g, h). The palate reconstructed by T M F was completely epithelialized at the 24 th week (Fig. 3a, b). A fine vascular plexus of low profile was noted below this repaired epithelium when compared with the prominent hairpin capillary loops in the normal palatal mucosa (Fig. 3c, d). The fine vascular plexus was separated from the T M F underneath by a welldefined collagen layer of less vascular density. The source of the vascular supply to the sub-epithelial plexus was consequently derived from the buccal and palatal sides of the reconstructed site, whereas the corresponding plexus on the normal side was directly supplied from below by the submucosal vascular network. Under scanning electron microscopy (SEM) with the selective removal of the epitheIium by corrosion, the sub-epithelial vascular plexus was observed as a fine interlacing network of capillaries

Microvascular density 50 40

~

: Fl:apc~2ntre -" I palate
=' -....

-

-

Flap margin

3O
20

10

2 4 6 8
1 I I I

12 14
I !

20
I

24
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Fig. 4. Comparison of the microvascular density between the flap centre, palatal wound margin and normal palate during the healing process of the temporalis flap.

The carotid arterial system of domestic cats has several characteristic features facilitating the infusion of the temporalis muscle. The external carotid artery is easily identified in the dissection of the neck. There is no confusion as to whether it is the external or internal carotid artery because the internal carotid artery in cats is normally degenerated to such an extent that an infusion medium such as lead oxide solution does not pass through 9. The largest branch of the external carotid artery in cats is the internal maxillary artery, from which branches supply the temporalis muscle. In cats, the internal maxillary artery forms a distinct arterial network in the infratemporal fossa area, called the rete mirabile or external rete, which is absent in humans 9. We found that the anterior deep temporal artery consistently branched out of the rete mirabile. The posterior deep temporal artery arose either from the rete mirabile or directly from the internal maxillary artery, whereas the posterior auricular artery consistently branched out of the internal maxillary artery directly. These 3 vessels were found to be the main vascular source of the temporalis muscle in cats, although FUJIMOTO 11 noted that additional minor vascular contributions may also arise from the buccal and superficial temporal arteries. The intramuscular vessel network and its extensive anastomosis in the temporalis muscle of cats has been defined by SAITO 14 and is comparable with the results presented. The vascular pattern on the normal hard palate has been extensively studied in mammals 12,13,~6,18,19. Although there are recognizable differences in the vascular network between different species, the blood supply is essentially derived from the greater palatine artery and forms different vascular layers in the submucosa and mucosa 13. The specific vascular pattern in the cat's palatal mucosa was published by TODATM and is in agreement with our findings of 2 distinct layers, the lamina propria and submucosal vascular networks. In a certain location of the hard palate, there was also a palatine venous plexus just beneath the sublnucosal vascular network. Notable capillary loops of hairpin configuration were projecting upward at right angles from the lamina propria vascular network found by our SEM

Microvasculatures of healing TMF
study. The configuration of these capillary loops was generally of simple hairpin shape as noted in animals, unlike the development of a more complex form in humans with triple loopings formed by additional secondary and tertiary capillaries19. The capillary loops were found to project into the mucosal papillae; LEE et al. 12 noted that the spacing, height and orientation of these loops closely correlated with the epithelial connective tissue interlace. On the hard palate, there were also welldeveloped rugae or plicae lying transversely in multiple rows formed by folding of the mucosa. These are considered important for the apprehension, transportation and mashing of food in animals. The capillary pattern immediately below these rugae was found to be more pronounced. In cats and dogs, the rugae have been noted to create undulation in the lamina propria vascular network when compared with other mammals ~3. Adequate information on two-dimensional changes of the vasculature in the reconstructed maxilla could be obtained from correlating the Indian ink specimens with histology. The changing microvascular pattern and vascular density of the surface layer corresponded well with the histological healing sequence. The vasculature of the healing muscle was at all times protected from the surface by an avascular layer before the epithelium was able to form a cover. On histology, this layer was made up of fibrin and inflammatory cells with eosinophilic staining. Since the deep temporal fascia has a blood supply distinct from that of the muscle, it may undergo ischaemic changes and form part of this avascular layer as well. However, the muscle remained viable and its intramuscular vessels well-perfused. These intramuscular vessels were confirmed to be the main source of vascular transformation on the surface layer of reconstruction. It was surprising to note that the normal side of the palate did not contribute significantly to the vascular changes in the healing flap. The vascular contribution of the muscle flap was gradually taken over by the vessels derived from the buccal side. When the temporalis was completely covered by mucosa at 24 weeks after surgery, the vascular source to the repaired mucosa became totally dependent on the palatal and buccal sides, with no contribution from the muscle. On histology, the repaired mucosa was shown to have different characteristics from the normal palatal mucosa. The normal microvascular layer and capillary loops were also confirmed to be quite different. This repaired mucosa was found to have a modified sub-epithelial vascular plexus with short capillary loops. The lack of prominent capillary loops correlates well with the deficient mucosal papilla and rugae in the repaired thin mucosa. STABLEINet al. 15 postulated that the capillary supply and its pattern might also be influenced by the thickness of the overlying epithelium. This reduced blood supply below the repaired mucosa explains the clinically pale-looking fibrotic mucosa that healed over the T M F in the long-term. An understanding of the microvascular changes of the healing temporalis flap not only complements the published histological changes6, but may have some clinical implications as well. The finding that a submucosal vascular plexus introduced by migrating epithelial cells forms the vascular supply to the repaired mucosa means that there is no fear of conducting secondary surgery on the healed TMF. The repaired mucosal flap can be raised from the muscle flap without running the risk of ischaemic necrosis. This enables bony reconstruction of the maxilla by a titanium tray supporting cortico-cancellous chips at a secondary stage for dental implant rehabilitation, similar to the primary reconstruction method published by our department 17. The space for the bony reconstruction is created by debulking of the muscle fibres and at the same time avoiding perforation of the repaired mucosa lining the maxillary sinus or nasal cavity. The repaired oral mucosal flap may then provide a cover over the tray, thus forming the oral barrier to facilitate bony healing.

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2. BRADLEYPE BROCKBANK J. The temporalis muscle flap in oral reconstruction - a cadaveric, animal and clinical study. J Maxillofac Surg 1981: 9: 139-45. 3. CHEUNGLK. The blood supply of the human temporalis muscle:a vascular corrosion cast study. J Anat 1996: 189: 4318. 4. CHEUNGLK. The vascular anatomy of the human temporatis muscle: implications for surgical splitting techniques. Int J Oral Maxillofac Surg 1996:25: 414~ 21. 5. CrmuNa LK. An animal model of maxillary reconstruction by the temporalis muscle flap. J Oral Maxillofac Surg 1996: 54: 1439-45. 6. CHEUNGLK. The epithelialization process in the healing temporalis myofascial flap in oral reconstruction. Int J Oral Maxillofac Surg 1997: 26:303 9. 7. CHEUNG LK, SAMMANN, TIDEMANH. Temporalis myofascial flap in maxillofacial reconstruction: clinical and histological studies of the oral healing process. Br J Oral Maxillofac Surg 1997: 35: 40612,
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MENEROB, SmRRAI. Temporalis myofascial flap for maxillofacial reconstruction. J Oral Maxillofac Surg 1991: 49: 106773. 9. DAVISDD, STORYHE. Carotid circulation in the domestic cat. Zool Series Field Museum Nat History 1943: 28: 547.

Acknowledgments'. This was part of a PhD
thesis of Hong Kong University and was supervised by Professor Tideman, whose help is gratefully acknowledged. The author would also like to thank the technicians of the Oral Bio-Science Laboratory and Dr. Nabil Samman for commenting on the manuscript. This investigationwas supported by the Hong Kong University CRCG research grant 337/253/0002.
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Professor L. K. Cheung Oral & Maxillofacial Surgery Prince Philip Dental Hospital 34 Hospital Road Hong Kong