Histological and molecular temporomandibular joint analysesafter mandibular advancement surgery: study in minipigsRicardo de Lima Navarro, DDS, MSc, PhD,a Paula Vanessa Pedron Oltramari, DDS, MSc,b

Eduardo Sant’Ana, DDS, MSc, PhD,c José Fernando Castanha Henriques, DDS, MSc, PhD,d

Rumio Taga, DDS, MSc, PhD,e Tânia Mary Cestari, BSc,f

Paulo César Rodrigues Conti, DDS, MSc, PhD,g Fernando Queiroz Cunha, MD, PhD,h andCarlos Ferreira Santos, DDS, MSc, PhD,i Bauru, São Paulo, BrazilUNIVERSITY OF SÃO PAULO

Objectives. The objective of this study was to elucidate the changes occurring in the temporomandibular joint (TMJ)after surgical mandibular advancement with different fixation techniques: bicortical screws (rigid fixation) andminiplates (semi-rigid fixation).Study design. Eighteen minipigs were equally and randomly divided into 3 groups: Group I (control), nonoperatedanimals; Group II, animals submitted to surgical advancement surgery and osteosynthesis by bicortical screws; andGroup III, animals submitted to surgical advancement surgery and osteosynthesis by miniplates. Four months after thesurgeries, the presence of interleukin (IL)-6 and IL-10 in synovial fluid samples was assessed in ELISA experiments.TMJs were histologically prepared.Results. Higher levels of IL-10 (P � .0436) were found for Group II. Descriptive histological analysis was compatiblewith the ELISA findings.Conclusions. Rigid fixation evokes more pronounced signs of bone remodeling in the TMJ, whereas malleable fixationpromotes a more intense inflammatory activity. Therefore, rigid fixation seems to transmit a higher impact of

postoperative masticatory forces to the TMJ. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2008;106:331-8)

The temporomandibular joint (TMJ) is a delicate struc-ture, through which other components of the mastica-tory system can generate, dissipate, and control jawmovements. Because of its characteristics as a livingtissue, when a stressful event persists for long enough,the TMJ has a tendency to adapt.

Interaction between surgery and orthodontics hasmade possible the treatment of severe malocclusions,also called dentofacial deformities (DDFS).1,2 Thetreatment for patients in such cases is normally aggres-sive, and although several techniques of mandibularosteotomies have been described in the past few de-

Bauru School of Dentistry, University of São Paulo, Brazil.aFormer Graduate Student, Department of Orthodontics.bGraduate Student, Department of Orthodontics.cAssociate Professor, Department of Stomatology.dProfessor, Department of Orthodontics.eProfessor, Department of Biological Sciences.fBiologist, Department of Biological Sciences.gAssociate Professor, Department of Prosthodontics.hProfessor, Department of Pharmacology, Ribeirão Preto MedicineSchool, University of São Paulo, Brazil.iAssociate Professor, Department of Biological Sciences.Received for publication Oct 8, 2007; returned for revision Jan 15,2008; accepted for publication Jan 23, 2008.1079-2104/$ - see front matter© 2008 Mosby, Inc. All rights reserved.

doi:10.1016/j.tripleo.2008.01.026

cades, only one is routinely performed by the greatmajority of oral maxillofacial surgeons, the bilateralsagittal split osteotomy (BSSO) technique.3-5

In the past, wires were widely used to stabilizeBSSO.6-8 Even though this technique offered satisfac-tory results, the possibility of movement between theproduced segments and consequent surgery failurecould be a problem. Stability was then achieved withthe introduction of rigid internal fixation (RIF)9 and, ascurrently referred to as semi-rigid internal fixation(SRIF).10-12

The different effects of each technique of BSSO on theTMJs are a common subject of interest in the litera-ture.13-19 RIF is the most resistant technique in mechanicaltests.20-23 In contrast, the great rigidity may cause torquein the condyle, moving it out of the glenoid fossa, andcausing transverse displacements of the proximal seg-ments.24 This kind of misplacement is not present whenmetal wires are used as fixation.25 A force when applied tothe condyle for long enough can overcome its adaptivecapacities and therefore induce bone remodeling, tem-poromandibular disorders (TMD), and also diminish thestability of the surgical results.15

The aim of this study was to evaluate the effects ofsurgical mandibular advancement on the TMJ of mini-pigs, by means of histological and molecular analysis.

For this purpose, 2 of the most used techniques were

331

OOOOE332 Navarro et al. September 2008

compared20,22: rigid internal fixation (bicortical screws)21

and semi-rigid internal fixation (miniplates).23,26

MATERIAL AND METHODSAnimals

Eighteen 15-month-old male minipigs (Minipig BR-1),27-30 weighing 35 kilograms each, were used. Theanimals were kept individually and fed with pig food(S4, Bravisco, Bastos-São Paulo, Brazil) in a dailyamount equivalent to 2% of the animal’s weight andwater ad libitum. The animals were randomly dividedinto 3 groups (6 animals each): Group I (control),nonoperated animals; Group II, animals submitted tosurgical advancement surgery and osteosynthesis bybicortical screws; and Group III, animals submitted tosurgical advancement surgery and osteosynthesis byminiplates.

MethodsThe institutional Ethics Committee for Research in

Animals approved the protocol of this study. Duringexaminations and surgical procedures the animals wereanesthetized with a combination of azaperone, Destress(Des-Vet) (1 mg/kg weight) and ketamine, Dopalen(Vetbrands, Jacareí-São Paulo, Brazil) (5 mg/kgweight).

All surgical interventions were performed by oneoperator (R.L.N.), who carried out the following se-quence. The surgical procedures began by the incisionof the labial commissure, to facilitate the access to themandibular posterior region, which was followed bythe intrabucal incision and the mucoperiosteal detach-ment. BSSO technique consisted of separating the man-dibular body segment of the 2 ascending mandibularramus, through parallel osteotomies, in the right andleft sides of the mandible.3-5 The osteosynthesis wasaccomplished only after the sides were completely sep-arated.

To carry out a standard advancement of 6 mm duringthe osteosynthesis, in both experimental groups, 2 chis-els with the respective thickness were used to stabilizethe proximal segment. The protocol to stabilize theproximal segment was manual pressure on the mandib-ular angle, which induces the condyle to assume acentric relation to the mandibular fossa. Concomitantly,the surgeon guided the segment intraorally to ensurethat it was properly positioned.15 Thus, in all fixationsites, 6-mm-chisels were placed with an orthogonalposition in relation to the mandibular bone surface,both in the superior and inferior aspects of the surgicalgap, and the fixation was performed. The perforationswere made under abundant irrigation and using thesuitable drills suggested by the manufacturer (MDT,

Rio Claro-São Paulo, Brazil). In Group II, 3 screws

(MDT) were installed with length compatible to thebone thickness and diameter of 2 mm, in an “L” in-verted position (Fig. 1). In Group III, two 2-mmminiplates (MDT) were used, one with 6 holes andanother with 4 holes, both with medium bridge (Fig. 2).After osteosynthesis, intra- and extraoral sutures wereperformed. Further, excursive mandibular movementswere tested to verify the resistance of the fixation, thebone positioning, and the occlusion.

After the surgical procedures, the following drugswere intramuscularly administered: antibiotic (Nuflor,Schering-Plough, Cotia-São Paulo, Brazil); corticoid(Naquasone, Schering-Plough); nonsteroidal anti-in-flammatory (Banamine, Schering-Plough) and analge-sic (D500, Pfizer), according to the veterinarian pre-scriptions.

After 120 days, the animals were humanely killedand the TMJs were dissected down to the articularcapsule, to permit the synovial fluid collection of theright and left TMJs of each animal. The collection wasperformed with the aid of a 3-mL syringe and a needleof 0.15 � 20.00 mm. The collected liquid was centri-fuged (Centrifuge Heraeus Sepatech Biofuge 15 R;Bensheim, Hessen, Germany; 10,000g, for 10 minutesat 4°C) and the supernatant was stored at �80oC, untilthe accomplishment of the enzyme-linked immunosor-bent assay (ELISA) test.31

After the synovial fluid collection, the areas of inter-est were harvested, dissected, and placed in 10% phos-phate-buffered formalin for 1 week. Thereafter thespecimens were decalcified in EDTA, dehydrated inethanol, clarified in xylol, and embedded in paraffin.Mesio-distal 5-�m sections of the central portion of

Fig. 1. Rigid internal fixation (bicortical screws): 3 screwswere installed with length compatible to the bone thicknessand diameter of 2 mm, in “L” inverted position.

each paraffin block were obtained and stained by he-

OOOOEVolume 106, Number 3 Navarro et al. 333

matoxylin-eosin method.32 The histological analysiswas blindly performed and the examiner observed thefollowing events: the presence of chronic inflammatoryinfiltrate, bone tissue remodeling and/or resorption, theintegrity of the capsular tissues (when available), thecondyle bone trabeculae, and the condyle surface car-tilage.

The supernatant was blindly analyzed by ELISAwith double ligant, as previously described,31 to quan-tify the presence of interleukin (IL)-6 (Pharmingen, St.Louis, MO, USA), a proinflammatory marker, andIL-10 (Pharmingen), an anti-inflammatory marker.33-35

The samples were thawed and diluted in growing con-centrations. Then, they were added to a 96-well plate,previously prepared with 100 �L/well of specific anti-body (Pharmingen) for the mentioned cytokines, inconcentration of 2 mg/mL (IL-6) and 1 mg/mL (IL-10)in buffered solution. The plate was stored at 4°C for 12hours (overnight). Washings were performed with buff-ered solution and incubated for 2 hours at 37°C in a 1%bovine serum solution.

Standard curves were built using standardizedamounts of IL-6 and IL-10 (Pharmingen) recombinantcytokines. Samples and control were applied in therespective wells and incubated overnight at 4°C. Theplates were washed carefully and the secondary biotin-ylated antibody (Pharmingen) was added. After 1 hour,the plate was washed 5 times with buffered solutionfollowed by the application of the avidin peroxidase(Sigma) compound diluted in 1:5000. After incubationfor 15 minutes, new wash was executed. Finally, O-phenylediamine (OPD, Dako, Glostrup, Denmark)chromogenic substrate was added (0.4 mg of OPD in

Fig. 2. Semi-rigid internal fixation (miniplates): two 2-mmminiplates were used, one with 6 holes and another with 4holes, both with medium bridge.

0.4 mL of H2O2 for each 1 mL of buffered solution).

After 15 minutes, the reaction was interrupted with 1MH2SO4 solution. The intensity of the color revealed bythe process was measured in spectrophotometer regu-lated in 490 nm, using a scanner for ELISA plates(Spectra Max 250, Molecular Devices, USES). Theresults were expressed in picogram (pg) of cytokine permL of synovial fluid.

Statistical analysisThe molecular analysis data obtained from the

ELISA test for the different groups were compared withanalysis of variance (ANOVA) and TUKEY post-hoctests, by using the SigmaStat software (SigmaStat,Windows 2.0; Jandel, San Rafael, CA, USA), with thelevel of significance set at 5%.

RESULTSDescriptive histological analysis

Group I presented characteristics of normality. Thearticular disk was composed of dense connective tissue,rich in collagen fibers, and with a thin aspect in thecentral area. The synovial membrane covered the inter-nal surface of the TMJ fibrous capsule and the marginsof the articular disk. The condyle showed a more reg-ular and symmetrical shape than in both experimentalgroups. The presence of areas with secondary condylarcartilage indicated that the animals were at the finalgrowth phase. The condyle presented as covered by alayer of fibrous tissue, below which a proliferate layerof nondifferentiated cells was detected. These cellsprobably originated the chondrocytes, which formedthe secondary articular cartilage (Fig. 3).

Similar articular disc characteristics to the controlgroup were found in Group II animals, except for themedial area, which was disorganized and with lowdensity. For this group, an asymmetrical condyle shapewas found when compared with Groups I and III,presenting a quite irregular surface and a discontinuoussecondary condylar cartilage. Additionally, the condylewas covered by a layer of fibrous tissue, with variedthickness and some areas of discontinuity. In the lateralregion, the most superior portion presented an increaseof the thickness, with bone remodeling of trabeculararrangement, which accompanied all the condylar area,from the central to the lateral portion. In addition, themedial area was more compact than in Group I, withextensive areas of resorption by osteoclasts (Fig. 4).

In Group III, the articular disk, the synovial mem-brane, and the condyle presented general characteristicssimilar to Group I. In the condyle, there were differentareas in relation to the control group, especially thecentral to lateral portion, where an increase of thethickness of the bone edge was demonstrated, with

bone remodeling of trabecular arrangement, which ac-

ue, fibr

OOOOE334 Navarro et al. September 2008

companied all the condylar area. In addition, the medialarea presented sites of resorption by osteoclasts. Cov-ering the bone tissue, there was a thin layer of connec-tive tissue richly cellularized and vascularized. Exter-nally, the fibrous capsule was disorganized, with somebunches of dense connective tissue, indicating its re-modeling (Fig. 5).

Molecular analysisThe ELISA test revealed similar levels of IL-6 be-

tween the groups, although Group III demonstratedhigher values of this proinflammatory marker (Table I).Furthermore, significantly greater levels of IL-10 (P �.0436) for Group II compared with the control group

Fig. 3. Coronal view of the condyle in Group I (control). A, Gmandibular condyle (CO), articular capsule (AC), lateral ptmedial region: pterigoideo muscle (PM), bone tissue (BT) surfof connective tissue (CT) rich in cells recovering the bone tiss

(Table II) were found.

DISCUSSIONThe present study demonstrated that there were his-

tological and molecular differences in the TMJs amongthe groups. Some authors described condylar alter-ations after surgeries for DDFS treatment.36-38 Clinicalsigns as dental and bone recurrences may occur de-pending on the severity of the alterations.15

The images obtained from the histological analysisdemonstrated characteristics of normality for the con-dyles of Groups I (control) and III (miniplates), al-though signs of bone remodeling were found only inGroup III samples. In Group II (bicortical screws), thecondyles presented irregular and asymmetrical shapes,with the presence of a discontinuous condylar cartilage

view showing the tissue distribution: intra-articular disc (ID),eo muscle (PM). B, Detail box magnification of condyle’sowing areas of resorption by osteoclasts (arrows), dense layerous capsule (FC) in the outer region (hematoxylin and eosin).

eneralerigoidace sh

and many secondary osteoclastic cells, which demon-

OOOOEVolume 106, Number 3 Navarro et al. 335

strated active bone resorption. The histological analysisdemonstrated that the technique used for Group IIcaused more pronounced condylar histological alter-ations than did the method used in Groups I and III.

When the ELISA test is considered, higher, nonsig-nificant, levels of IL-6 were found for Group III. Yet,the detection of lower levels of IL-10 for this groupsuggests the presence of an active inflammatory pro-cess.33-35 For Group II, ELISA test demonstrated sta-tistically significantly higher levels of IL-10 than in thecontrol group. Additionally, lower levels of IL-6 weredetected, which suggested the presence of previousinflammatory activity that was diminished or termi-nated after the experimental period (120 days).

Previous studies had already tested markers withELISA for synovial fluid, but only to investigate

Fig. 4. Coronal view of the condyle in Group II (Rigid interdistribution: intra-articular disc (ID), mandibular condyle (Ccondyle’s medial region; bone tissue (BT), with extense area(FT) similar to fibrous cartilage (hematoxylin and eosin).

TMDs.39,40 Further investigations relating DDFSs or

other types of fixations for orthognathic surgery are stillnecessary to elucidate the real role of the surgery tech-nique on TMJ alterations. Additional time course sur-gical analyses would be necessary to more preciselydefine the duration of the inflammatory activity in theTMJs. Differences between the 2 types of fixation,however, are suggested to occur after the experimentalperiod of 120 days.

Based on that and considering the histological andmolecular analyses, it can be inferred that condyles ofGroup III presented some degree of inflammation, al-though not severe enough to cause significant alteration incondyle shape. On the other hand, condyles of Group IIpresented alterations in the form and signs of previousinflammation, suggesting a more intense inflammatorypeak than in Group III, which was probably responsible

tion, bicortical screws). A, General view showing the tissueeral pterygoid muscle (PM). B, Detail box magnification ofsorption by osteoclasts (arrows), recovered by fibrous tissue

nal fixaO), lats of re

for the condylar remodeling found in this group.

), indi

OOOOE336 Navarro et al. September 2008

Many authors demonstrated the efficacy of rigid fix-ation in the resistance aspect.20-23,26,41 Its superiority in

Fig. 5. Coronal view of the condyle in Group III (semi-rigiddistribution: intra-articular disc (ID), mandibular condyle (CObox magnification of condyle’s medial region: bone tissue (Bbone tissue there is a dense connective tissue (CT) layer rich ifibrous capsule (FC), with some remaining bunches (asterisk

Table I. Concentration of IL-6 (�g)/mL in groups I, II,and III: mean, standard error, analysis of variance

IL-6 Mean Standard error P

Group I 83.22 14.65 .4960Group II 103.52 38.23Group III 140.05 37.65

IL, interleukin.

rigidity and, when well executed, in immediate posi-

tional stability is undeniable. The possible effects onthe TMJs, however, must be emphasized.15 Arnett,15 in1993, explained the possible failures of the techniqueconsidering the condylar positioning. These failuresmay happen when any internal fixation method is used;however, bicortical fixation does not present reversiblecharacteristics.23

The semi-rigid fixation technique is efficient becauseof its reliable feasibility, which depends directly onsurgeon training, reduced surgical time, and, especially,diminished risks of condylar problems.15

Similar results to those found in this study were

al fixation, miniplates). A, General view showing the tissueular capsule (AC), lateral pterigoideo muscle (PM). B, Detailh areas of resorption by osteoclasts (arrows). Recovering theand little blood vessels (blank arrow). Outer, a desorganized

cating capsule remodeling (hematoxylin and eosin).

intern), articT) witn cells

reported by Ellis and Hinton42 in 1991. Their conclu-

OOOOEVolume 106, Number 3 Navarro et al. 337

sions, however, were based only on lateral cephalomet-ric radiographs and histological tests. These authorsdemonstrated the presence of significant condylar pos-terior resorption when rigid fixation was used comparedwith the semi-rigid technique. The authors did notanalyze different types of rigid fixation, or use morespecific methods, as the ELISA test applied in ourmethodology. However, their results showed signs thatcondylar resorption may occur due to condylar retru-sion.

Based on the present results, miniplates with mono-cortical screws are efficient to support the masticatoryforces and to partly transmit them to the TMJs. On theother hand, bicortical screws seem to transfer higherlevels of loading to the TMJs. There is a serious con-cern about the possible mandibular retrusion after sur-gical mandibular advancement. This later posterior mi-gration15 could be a consequence of the condylarcompression and was observed in the histological slidesof this work.

Regardless of the technique used, mandibular posi-tion changes after surgical advancement surgery cangenerate condylar alterations.15,43,44 However, the lit-erature15,16,45,46 demonstrates that the postsurgical con-dylar alterations are more evident when rigid fixation isused, as it was revealed in this study.

CONCLUSIONSConsidering the methodology used, the results indi-

cated that rigid fixation (bicortical screws) resulted inhigher levels of condylar remodeling, but with indica-tions of a less intense inflammatory process, whencompared with the semi-rigid fixation technique(miniplates). Therefore, the use of rigid fixation seemsto transmit higher impact of the masticatory forces tothe TMJs than semi-rigid fixation. Further clinical stud-ies are necessary to confirm these data.

The authors express their gratitude to Giuliana BertoziFrancisco for her excellent technical assistance in theELISA experiments. We also thank MDT for kindlyproviding the fixation material.

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Table II. Concentration of IL-10 (�g)/mL in groups I,II, and III: mean, standard error, analysis of variance

IL-10 Mean Standard error P

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Group II 138.91b 14.26Group III 95.64ab 14.74

Different letters represent statistically significant differences.IL, interleukin.

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Reprint requests:

Ricardo de Lima Navarro, DDS, MSc, PhDRua Júlio Maringoni, 6-17Vila Nova Santa ClaraBauru, SP17014-050 Brazil

r-navarro@hotmail.com