microvascular healing over temporalis

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    4 7 0 Cheung

    infusion media: Indian ink solution, lead ox-ide solution or methyhnethacrylate resin.The composi t ion of the infus ion media andthe methods of prep ara t ion of the maxi llaryspecimens have been reported previously4.

    The cats were randomly assigned into oneof 3 groups and the choice of infusion me-

    dium was made according to the defined sac-rifice time for that group. The lead oxidesolution was selected to i l lustrate the arterialpat tern in the TMF fol lowing i t s t ransferinto the oral cavity for maxillary reconstruc-t ion. The In dian ink solut ion would demon-strate the microvascular network and thechanges of the TMF dur ing the heal ing pro-cess. The methylmethacrylate resin wouldform vascular corros ion cas ts to complementthe result of the Indian ink findings and toillustrate the microvasculatures in three di-mensions underneath the repai red ora l mu-cosa and compared wi th the contra la tera lnormal palatal mucosa. The lead oxide in-

    fused head specimens were radiographed(Hewlett Packard x-ray system 43805N,USA) at 70 kvp for 2-5 minutes. The ex-posure t ime was adjusted according to t ire

    thickness of the specimens and the imageswere captmzd using instant fi lms (Polaroid550 positive & negative films, USA). The In-dian ink infused specimens were embedded inparaffin blocks and prepared into thin sec-

    tions of 10/~m thickness and thick sectionsof 300 #m thickness. The thin sections weres ta ined wi th haematoxylin and eos in (H&E)and the thick sections remained unstained;bo th were mo unt ed o n glass slides. Th e speci-

    Fig. 1. Macrovascular network changes fol-lowing temporalis flap transposition to max-ilia by lead oxide infusion. On the radio-graphic submental vertex view following re-moval of the mandible in cat, the right side(R) was the normal and the left (L) was theoperated side. On the normal side, the pathof the internal maxillary artery is i l lustratedwith its branches supplying the maxilla: (a)internal maxillary artery, (b) infra-orbital ar-tery, (c) posterior palatine artery, (d) de-scending palatine artery, and (e) sphenopa-latine artery. However, on the operated side,obvious medial displacement of the internalmaxillary artery by the muscle was noted.

    Fine branches derived directly from the retemirabile (*) and the inte rnal maxillary arterywere seen.

    Fig. 2. Microvascular changes in the superficial layer of the tempo ratis flap during th e healingprocess. A: In the acute inflammatory phase during the first 6 weeks, the microvasculaturesof the superficial layer were dilated, formin g bush-like bundles. The vascu lar con tribut ion tothis layer was derived from the muscle flap rather than the normal side of the palate (Indianink x20). B: Histological section of the corresponding palate/flap junction as in A. (H&E 50). C: In the chronic inflamm atory phase durin g the 8th-14 th weeks, the dilated microvas-cular bundles abov e the temporalis m uscle fibres became more discrete but sti l l separated fromthe surface by an avascular layer. New microvasculatures (I~) were introduc ed by the migratingepithelium from the palatal side (Indian ink D: Histological section of similar site as inC, showing onset of chronic inflamm atory phase on the flap (H&E E: At higher magni-fication of the advanc ing 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, showingepithelial migrat ion an d hy perplastic changes (H&E ?

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    mens were examined under light microscopy(Leitz Orthoplan, Germany). The corrosionvascular casts infused by methylm ethacrylateresin were sectioned into small seganents andmounted on copper stubs with conductivecolloidal carbon. The casts were sputtercoated with gold and examined under scan-

    ning electron microscopy (Joel JXA-840SEM, Japan).

    In addition to the morphological descrip-tion o f the microvascular networks at the dif-ferent healing times, morphometric analysisof the vascular density in the Indian ink in-fused thick sections was performed. The im-ages of the vascular networks were capturedthrough a video camera (JVC TK-1280E, To-kyo, Japan) mounted on the microscope (Ni-kon Ophtihot, Japan) to a computerized im-age analyzer system (Leica Quantimet 500+,Cambridg e, England). A standard field wascreated and the microvascular networkswithin this field were quantified as the vascu-

    lar density percentage. The vascular densityat the wound margin, wound centre andcontralateral normal palatal mucosa wasmeasured 5 times from separate slides at eachof the sacrifice times. The means and stan-dard deviations of the vascular density per-centage of the three selected fields were com-pared by one-way analysis of variance(ANOVA). If the P v alue was less than 0.05,then further comparison of the groups wasconducted by a Tukey-Kramer multiple com-parison test to identify the significant pairs.Instat software (Instat Ver 3.0, Graph PadSoftware Ltd., S an Diego, USA ) was used forthe statistical analysis.

    Microvasculatures of healing TM F 471

    R e s u l t s

    Wi t h t h e T M F t r a n s p o se d i n t o th e o ra lcav i ty, t he mos t obv ious change was them e d i a l d i s p l a c e m e n t o f t h e i n t e r n a lmax i l l a ry a r t e ry by the musc le bu lk(F ig . 1). The no rm a l a r r ang emen t o fthe an te r io r pa l a t ine a r t e ry and i t sb ranches d i sappea red and was r ep lacedby f ine b ranches de r ived f rom the r e t emi rab i l e o r d i r ec t ly f rom the in t e rna lmax i l l a ry a r t e ry fu r the r back . Th i s i n -d i c at e s t h e m a i n t e n a n c e o f m a c r o v a s c u -l a r s up p l y t o t h e T M F f r o m i ts o r i gi n a lsou rce wi th no s ign i f i can t a l t e r a t iondur ing the hea l ing phases . The musc lef ib re s r ema in ed w e l l pe r fused by mic ro -vesse l s a s con f i rmed by the Ind ian inkin fus ion o f t he fib res . The hea l ing T M Fwas sh i e lded f rom the o ra l cav i ty by ath i ck l aye r o f vascu la r t i s sue a t a l l t imesdur ing the hea l ing p rocess . Th i s supe r-f i c i a l vascu la r l aye r was found tochange i t s pa t t e rn and dens i ty, co r r e l a t -ing wi th the d i f f e ren t phases o f h i s to -

    log ica l hea l ing .I n t h e a c u t e i n f l a m m a t o r y p h a s e o fthe TMF hea l ing du r ing the f i r s t 6

    Fig. 3. Com pariso n o f sub-epithelial microvascular plexus between the repaired mucosa andnorm al palat al muco sa at 6 month s postoperatively. A: The tempo ralis muscle remained well-perfused and was covered by a flat mucosa (Indian ink ?420). B: The microvascular networkon the no rma l side of hard palate (Indian ink ?

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    472 C h e u n ge r a ll y, w i t h n o a p p a r e n t c o n t r i b u t i o nf r o m t h e p a l a t a l s i d e . Wi t h t h e p r o -gress ive change in to the chron ic in -f l a m m a t o r y p h a s e d u r i n g t h e 8 t h - 1 4 t hw e e k s , t h e d i l a t e d v a s c u l a r b u n d l e sa b o v e t h e t e m p o r a l i s m u s c l e f i b r e s b e -

    c a m e m o r e d i s c r e te b u t w e r e s ti l l s e p a r -a t e d f r o m t h e s u r f a c e b y a n a v a s c u l a rl a y e r. F o r m a t i o n o f a n e w v a s c u l a rp l e x u s w a s n o t e d b e l o w t h e a d v a n c i n ge p i t h e l i u m a t t h e p a l a t a l a n d b u c c a ls i d es o f t h e f l a p ( F i g . 2 c - f ). A t t h e1 8 hweek, the vesse l s in the super f ic ia l vas -c u l a r l a y e r h a d b e c o m e l e s s d e n s e a n ds m a l l e r i n d i a m e t e r, w h i l e t h e f l a p w a sp r o g r e s s i n g t h r o u g h t h e p r o l i f e r a t i v ep h a s e o f h e a l i n g ( F i g . 2 g , h ).

    T h e p a l a t e r e c o n s t r u c t e d b y T M Fw a s c o m p l e t e l y e p i t h e l ia l i z e d a t t h e 2 4 hweek (F ig . 3a , b ) . A f ine vascu la r p lexuso f l o w p r o f i l e w a s n o t e d b e l o w t h i s r e -p a i r e d e p it h e li u m w h e n c o m p a r e d w i t ht h e p r o m i n e n t h a i r p i n c a p i l l a r y l o o p s i nt h e n o r m a l p a l a t a l m u c o s a ( F i g . 3 c , d ) .T h e f i n e v a s c u l a r p l e x u s w a s s e p a r a t e df r o m t h e T M F u n d e r n e a t h b y a w e l l -d e f i n e d c o l l a g e n l a y e r o f l e s s v a s c u l a rd e n si t y. T h e s o u r c e o f t h e v a s c u l a r s u p -p l y t o t h e s u b - e p i t h e l i a l p l e x u s w a sc o n s e q u e n t l y d e r i v e d f r o m t h e b u c c a la n d p a l a t a l s i d e s o f t h e r e c o n s t r u c t e ds i t e , w h e r e a s t h e c o r r e s p o n d i n g p l e x u so n t h e n o r m a l s i de w a s d i r e c tl y s u p -

    p l i e d f r o m b e l o w b y t h e s u b m u c o s a lv a s c u l a r n e t w o r k .U n d e r s c a n n i n g e l e c t r o n m i c r o s c o p y

    ( S E M ) w i t h t h e s e le c ti v e r e m o v a l o f t h ee p i t h e I i u m b y c o r r o s i o n , t h e s u b - e p i -t h e l i a l v a s c u l a r p l e x u s w a s o b s e r v e d a sa f i n e in t e r l a c i n g n e t w o r k o f c a p i l l a r i e s

    c o n n e c t e d

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