- Email: [email protected]
Impact of Temporomandibular Joint Discectomy on Condyle Morphology: An Animal Study
Accepted Manuscript Impact of temporomandibular joint discectomy on condyle morphology: an animal study Reinaldo Abdala-Júnior, DDS, MS (Graduate student), Arthur Rodriguez Gonzalez Cortes, DDS, PhD (Postgraduate fellow), Eduardo Massaharu Aoki, DDS , MS (Graduate student), Simone Ferreira, PhD (Researcher), João Gualberto Cerqueira Luz, DDStabe, PhD (Associate professor), Emiko Saito Arita, DDS, PhD (Associate professor), Jefferson Xavier de Oliveira, DDS, PhD (Associate professor) PII:
S0278-2391(17)31544-6
DOI:
10.1016/j.joms.2017.12.019
Reference:
YJOMS 58101
To appear in:
Journal of Oral and Maxillofacial Surgery
Received Date: 12 September 2017 Revised Date:
20 December 2017
Accepted Date: 20 December 2017
Please cite this article as: Abdala-Júnior R, Gonzalez Cortes AR, Aoki EM, Ferreira S, Cerqueira Luz JG, Arita ES, Xavier de Oliveira J, Impact of temporomandibular joint discectomy on condyle morphology: an animal study, Journal of Oral and Maxillofacial Surgery (2018), doi: 10.1016/ j.joms.2017.12.019. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Impact of temporomandibular joint discectomy on condyle morphology: an animal study Reinaldo Abdala-Júnior, DDS, MS (Graduate student)1
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Arthur Rodriguez Gonzalez Cortes, DDS, PhD (Postgraduate fellow)1,2,3 Eduardo Massaharu Aoki, DDS , MS (Graduate student)1 Simone Ferreira, PhD (Researcher)4
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João Gualberto Cerqueira Luz, DDStabe, PhD (Associate professor)4 Emiko Saito Arita, DDS, PhD (Associate professor)1
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Jefferson Xavier de Oliveira, DDS, PhD (Associate professor)1
1
Oral Radiology Division, School of Dentistry, University of Sao Paulo, Brazil Martinos Center for Biomedical Imaging, Department of Radiology, Massachusetts General Hospital, Charlestown, MA 02129, USA 3 Harvard Medical School, Boston, MA 02115, USA 4 Department of Oral Surgery, School of Dentistry, University of Sao Paulo, Brazil
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Corresponding author: Dr. Reinaldo Abdala Júnior , Department of Stomatology, School of Dentistry, University of São Paulo, Av. Lineu Prestes, 2227. Zip code: 05508-000. São Paulo, SP, Brazil. Telephone and fax number: +55 11 3091-7831. E-mail address: [email protected].
Running Title: Impact of discectomy on condyle morphology
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Abstract
Purpose: Temporomandibular disorders (TMD) lead to parafunctional activity
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that may alter bone remodeling of mandibular components. This animal study aimed to assess the impact of temporomandibular joint discectomy on condyle bone microarchitecture.
SC
Material and Methods: A total of 30 one-month-old Wistar rats were assessed and divided in three equal groups (two tests and one control) of 10. The first test
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group underwent disc removal (DRm; the second test group underwent disc and condylar cartilage removal (DCR); and the 10 remaining rats were analyzed as sham-operated controls (CTR), following a split-mouth design. Rats were sacrificed two months after surgery, and the respective mandibles were scanned
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with micro-CT for quantitative morphometric analysis.
Results: There were significant differences among the three groups analyzed (DR, DCR and CTR) for Bone volume fraction (BV/TV, p=0.044), Structure model
EP
index (p<0.001), Fractal Dimension (p=0.024) and porosity (p=0.023). In addition, operated and contralateral non-operated sides significantly differed for all
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variables in at least one of the test groups (p<0.05), but not in the control group (p>0.05).
Conclusion: Within the limitations of this study, the present results suggest that discectomy may lead to alterations of the mandibular condylar morphology.
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Keywords: Temporomandibular joint; temporomandibular disorders; micro-
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computed tomography, alveolar bone; Mandibular condyle.
Introduction
It is known that parafunctional activity may lead to morphological changes
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of maxillofacial hard and soft tissues.[1-3] Such conditions can be triggered by alterations of temporomandibular joint (TMJ) components, which may have a
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significant impact on mandibular structures such as the condyle.[1] Previous studies observed that TMJ disk integrity contributes to the healing process in cases of condyle fracture.[4, 5] Temporomandibular
disorders
(TMD)
involve
alterations
of
joint
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components that can be treated with several methods, such as physical therapy, occlusal adjustment, medications or surgeries.[6] One of the surgical modalities that has been used to treat painful TMD cases not responding to non-surgical
EP
treatment is the discectomy. Although this is a predictable treatment modality, it may lead to postoperative clinical conditions such as condyle flattening and
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sclerosis.[7, 8]
Despite of the above-mentioned evidences, little is known on the
microstructural effects of discectomy on condyle bone morphology. In this context, micro-computed tomography (µCT) has been used to detect alterations of micro-architecture of rat trabeculae in images with adequate isotropic resolution. [9]
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Thus, the aim of this study was to assess the impact of TMJ discectomy
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on the condyle bone microarchitecture of rats.
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Study Sample
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Material and Methods
A total of 30 one-month-old male Wistar rats (average weight: 70.1±3.6 grams) were included in this study. All rats received a diet based on granulated commercial rodent (Labina, Agribrands-Purina Ltda, Paulinia, Brazil) and water
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ad libitum. The handling of animals was in accordance with the ethical principles proposed by the Brazilian College of Animal Experimentation. Approval was obtained from the animal ethics, care and use committee of the university of this
EP
study (protocol number: 035-2015).
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Groups’ definition
Rats were classified into three equal groups (disc removal (DR); disc and
condylar cartilage removal (DCR); and sham-operated controls (CTR)), following a split-mouth design:
In the DR group: rats undergoing disc removal surgery in one side, and no surgery in the contralateral side (n=10); in the DCR group: Rats undergoing both
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disc and condylar cartilage removal in one side, and no surgery in the contralateral side (n=10); in the CTR group: Rats that underwent sham surgery in
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one side, and no surgery in the contralateral side (n=10).
Surgical procedures
All procedures were performed under general anesthesia induced by
SC
xylazine hydrochloride 10 mg/kg (Rompum, Bayer, Porto Alegre, RS, Brazil) and ketamine hydrochloride 25 mg/kg (Dopalen, Vetbrands, Paulínia, SP, Brazil).
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The present study followed a previously described methodology for conducting split-mouth design studies on local interventions in the TMJ.[5, 10-12] Only the right side of all rats underwent surgical interventions while the left side remained intact. Surgical flaps were opened with a one-centimeter-long
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horizontal incision that followed the extension of the zygomatic arch, following its identification by palpation.
Each group underwent only one surgical intervention, described as
EP
following: in the CTR group, flaps were secured with no actual intervention on the TMJ (sham-operated). In the DR group, the articular disc was removed using
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scalpel and straight forceps. Finally, in the DCR group, rats underwent both articular disc and condylar cartilage removal. The latter was carefully performed using a specific scalpel blade (# 15). Following the above-mentioned procedures, flaps were secured by layers
with monofilament sutures in all groups. After a two-month postoperative follow-
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up, all rats were sacrificed by lethal dose of anesthesia. Then, the heads were
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removed, fixed in formalin and dissected.
Micro-CT
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Multiplanar and volumetric reconstructions of the condyle bone of all specimens were obtained using a Micro-CT system - SkyScan Model 1176
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(Skyscan; Kontich, Belgium) with the following imaging protocol: 50 kVp, 500 mA, 16 bits, 8.71µm of resolution, 0.5 degree step of the rotation angle and 360 degrees of rotation. The specimens were mounted on a low-density foam base and fixed by a PVC film (Polyvinyl Chloride) before the scan. Projections and
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volumetric images were reconstructed using NRecon software (Bruker, Belgium). A volume of interest [13] enclosing the entire condyle area was delineated and considered in all analyses of trabecular bone micro-architecture. To ensure
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uniformity of the VOI selection, the hemimandibles had been previously aligned on the X, Y and Z in the Data Viewer software (Bruker, Belgium). Then, 2D and
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3D morphometric analysis were performed using Skyscan CT-Analyser software (Bruker, Belgium).
The following parameters of trabecular microstructure were quantitatively
assessed: bone volume fraction (BV/TV), which is the ratio of bone volume to the total volume of the sample, and has been considered a measure of strength and stiffness of cancellous bone;[14] trabecular separation (Tb.Sp), which is also
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related to bone strength and is usually altered with age;[15] fractal dimension (FD), which is a statistical texture analysis associated with bone stiffness;[16] connectivity density (Conn.Dn), which is a predictor of vascular network;[17] total
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porosity; and structure model index (SMI), which normally varies between 0, for perfect plates, and 3, for perfect rods. Negative SMI values are associated with
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isolated marrow spaces.
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Statistical analysis
The sample size used was determined considering an effect size of f = 0.6, to give the study a power of 80% (type II β error = 0.2) at a significance level of 5% (type I α error = 0.05), resulting in a total sample size of 30 rats, equally
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distributed into three groups. Normality was calculated for all outcome variables (BV/TV, Tb.Sp, FD, Conn.Dn, porosity and SMI) with the Shapiro-Wilk test. Paired Student's t-test was used to compare the variables between left and right
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sides of the same mandibles (i.e within-subject measurements). In addition, results for the right (operated) side of all groups (DR, DCR and CTR) were
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compared (i.e. between-subject measurements) with analysis of variance test (One-way ANOVA). Post-hoc analysis was conducted with Tukey HSD. Because of our split-mouth study design, the aforementioned analyses did not need to be adjusted to confusion variables such as age and gender. A 5% significance level for all parameters (p <0.05) was adopted. The software used for statistical analysis was SPSS (IBM SPSS Version 20.0 Chicago, IL, USA).
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Results
Normality was confirmed for all variables (p>0.05). Regarding within-
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subject measurements, operated (Figures 1, 2 and 3) and contralateral nonoperated sides significantly differed for all significant variables in at least one of the test groups. For BV/TV (Figure 4), both DR and DCR differed between sides
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(p=0.031 and p=0.019, respectively). For FD (Figure 5), total porosity (Figure 6), and SMI (Figure 7), only the DCR group showed significant differences (p<0.001,
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p=0.003, and p=0.001, respectively). No significant differences between sides were observed in the control group (p>0.05).
Furthermore, there were significant between-subject differences among the three groups analyzed (DR, DCR and CTR) for BV/TV (p=0.044), SMI (p<0.001), FD (p=0.024) and porosity (p=0.023). All aforementioned differences
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were confirmed by Tukey’s Post-hoc test (p<0.05). No other variables differed
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Discussion
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significantly among groups (p>0.05).
Discectomy leads to morphologic alterations of the mandibular condyle,
which may have an impact on occlusal function. In order to adapt the bone to the new mechanical demands, increased mechanical loads will lead to new bone formation and remodeling. Similarly, a lack of mechanical loads might lead to low bone trabecular and mineral density.[18, 19]
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In the present study, discectomy and subsequent cartilage removal led to worsen morphometric conditions of the condyle. TMJ stability depends on a muscular condition that is achieved when the condyles are located in their most
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superoanterior position regarding the articular fossae, with the disc joint itself interposed between the aforementioned two structures.[20] In addition, TMD has been found to alter jaw muscles properties, such as functional efficiency, force of
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contraction and electrical activity, leading to lack of stimulation on the condyle bone.[21] Our hypothesis that the DCR group would present worsen
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morphometric conditions than both DR and CTR groups was confirmed, supporting previously described evidences.[22] In addition, DCR groups presented significant within-subject differences in more morphometric variables than DR and CTR groups. The latter, in particular, did not present any significant
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differences between operated and non-operated sides. Such findings are in agreement with a previous similar study in the literature.[12] On the other hand, there was no significant difference in some of the
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morphometric parameters, such as trabecular separation, among the three groups. This could indicate the occurrence of a compensatory mechanism of
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bone morphometry triggered by the lack of condylar cartilage due to the great adaptive capacity of immature TMJ.[5, 23] One limitation of this study, however, is that no direct assessments of
muscular activities could be performed for the groups analyzed. Future clinical and animal studies monitoring of the activity of jaw muscles would be recommended to understand compensatory growth mechanisms involving the
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TMJ, taking into consideration that mandibular growth is directly affected by muscular activity.[3, 19, 24] Little is known on the effects of TMJ alterations on mandibular bone sites.
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To our knowledge, this is the first study addressing the effect of discectomy on the condyle bone morphometry, offering microscopic evidences that support studies that found clinical alterations in cases of discectomy, such as condyle
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flattening and sclerosis.[7, 8] Micro-CT has been considered the best option to quantifying 3D bone microarchitecture, allowing for obtaining direct volumetric
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and morphometric measurements of trabecular bone features, contrasting with 2D histological assessment based on stereological methods.[9, 25, 26] Furthermore, micro-CT is the most used method to assess bone morphometric variables. Laboratorial assessments based on finite element analyses concluded
properties.[27-29]
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that trabecular microstructure has a significant impact on bone biomechanical
In conclusion, within the limitations of this study, the present results
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suggest that either removal of the articular disk or concomitant removal of both disc and articular cartilage may lead to worsen condyle bone morphometric
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conditions, as compared to the contralateral non-operated sides.
Acknowledgements: The authors declare that there are no conflicts of interest in this study and that they agree and have viewed this submission. Two master scholarships were granted to RAJ and EMA by the Coordination for the Improvement of Higher Education Personnel (CAPES – Brazil). A post-doctoral scholarship was granted to ARGC by the National Council for Scientific and Technological Development (CNPq)—Science without Borders, Brazil, no. 232643/2013-0.
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REFERENCES
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1. Hu Y, Yang HF, Li S, Chen JZ, Luo YW, Yang C: Condyle and mandibular bone change after unilateral condylar neck fracture in growing rats. Int J Oral Maxillofac Surg 41:912, 2012 2. Schellhas KP, Pollei SR, Wilkes CH: Pediatric internal derangements of the temporomandibular joint: effect on facial development. Am J Orthod Dentofacial Orthop 104:51, 1993 3. Cortes AR, Jin Z, Morrison MD, Arita ES, Song J, Tamimi F: Mandibular tori are associated with mechanical stress and mandibular shape. J Oral Maxillofac Surg 72:2115, 2014 4. Li Z, Zhang W, Li ZB, Li JR: Mechanism in favorable prognosis of pediatric condylar fractures managed by closed procedures: an experimental study in growing rats. Dent Traumatol 26:228, 2010 5. Luz JG, de Araujo VC: Rotated subcondylar process fracture in the growing animal: an experimental study in rats. Int J Oral Maxillofac Surg 30:545, 2001 6. Tanaka E, Detamore MS, Mercuri LG: Degenerative disorders of the temporomandibular joint: etiology, diagnosis, and treatment. J Dent Res 87:296, 2008 7. Eriksson L, Westesson PL: Discectomy as an effective treatment for painful temporomandibular joint internal derangement: a 5-year clinical and radiographic follow-up. J Oral Maxillofac Surg 59:750, 2001 8. McKenna SJ: Discectomy for the treatment of internal derangements of the temporomandibular joint. J Oral Maxillofac Surg 59:1051, 2001 9. Bouxsein ML, Boyd SK, Christiansen BA, Guldberg RE, Jepsen KJ, Muller R: Guidelines for assessment of bone microstructure in rodents using microcomputed tomography. J Bone Miner Res 25:1468, 2010 10. Cavalcanti SC, Correa L, Luz JG: Facial symmetry evaluation after experimentally displaced condylar process fracture in methotrexate treated rats. Acta Cir Bras 27:210, 2012 11. Cavalcanti SC, Correa L, Mello SB, Luz JG: The effect of methotrexate on the bone healing of mandibular condylar process fracture: an experimental study in rats. J Craniomaxillofac Surg 42:1133, 2014 12. Toledo LG, Cavalcanti SC, Correa L, Luz JG: Effects of injury or removal of the articular disc on maxillomandibular growth in young rats. J Oral Maxillofac Surg 72:2140, 2014 13. Merrem A, Hofer S, Voit D, Merboldt KD, Klosowski J, Untenberger M, Fleischhammer J, Frahm J: Rapid Diffusion-Weighted Magnetic Resonance Imaging of the Brain Without Susceptibility Artifacts: Single-Shot STEAM With Radial Undersampling and Iterative Reconstruction. Invest Radiol 52:428, 2017 14. Nazarian A, von Stechow D, Zurakowski D, Muller R, Snyder BD: Bone volume fraction explains the variation in strength and stiffness of cancellous bone affected by metastatic cancer and osteoporosis. Calcif Tissue Int 83:368, 2008
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15. Stauber M, Muller R: Age-related changes in trabecular bone microstructures: global and local morphometry. Osteoporos Int 17:616, 2006 16. Wu Y, Adeeb S, Doschak MR: Using Micro-CT Derived Bone Microarchitecture to Analyze Bone Stiffness - A Case Study on Osteoporosis Rat Bone. Front Endocrinol (Lausanne) 6:80, 2015 17. Zhao F, Liang J, Chen X, Liu J, Chen D, Yang X, Tian J: Quantitative analysis of vascular parameters for micro-CT imaging of vascular networks with multi-resolution. Med Biol Eng Comput 54:511, 2016 18. Frost HM: A 2003 update of bone physiology and Wolff's Law for clinicians. Angle Orthod 74:3, 2004 19. Frost HM: Bone's mechanostat: a 2003 update. Anat Rec A Discov Mol Cell Evol Biol 275:1081, 2003 20. Okeson JP: Evolution of occlusion and temporomandibular disorder in orthodontics: Past, present, and future. Am J Orthod Dentofacial Orthop 147:S216, 2015 21. Liu ZJ, Yamagata K, Kasahara Y, Ito G: Electromyographic examination of jaw muscles in relation to symptoms and occlusion of patients with temporomandibular joint disorders. J Oral Rehabil 26:33, 1999 22. Tingey TF, Shapiro PA: Selective-Inhibition of Condylar Growth in the Rabbit Mandible Using Intra-Articular Papain. American Journal of Orthodontics and Dentofacial Orthopedics 81:455, 1982 23. Dimitroulis G, Slavin J: The effects of unilateral discectomy and condylectomy on the contralateral intact rabbit craniomandibular joint. J Oral Maxillofac Surg 64:1261, 2006 24. Yonemitsu I, Muramoto T, Soma K: The influence of masseter activity on rat mandibular growth. Arch Oral Biol 52:487, 2007 25. Particelli F, Mecozzi L, Beraudi A, Montesi M, Baruffaldi F, Viceconti M: A comparison between micro-CT and histology for the evaluation of cortical bone: effect of polymethylmethacrylate embedding on structural parameters. Journal of Microscopy 245:302, 2012 26. Feldkamp LA, Goldstein SA, Parfitt AM, Jesion G, Kleerekoper M: The direct examination of three-dimensional bone architecture in vitro by computed tomography. J Bone Miner Res 4:3, 1989 27. Caldwell CB, Moran EL, Bogoch ER: Fractal dimension as a measure of altered trabecular bone in experimental inflammatory arthritis. J Bone Miner Res 13:978, 1998 28. IChappard D, Basle MF, Legrand E, Audran M: Trabecular bone microarchitecture: a review. Morphologie 92:162, 2008 29. Prot M, Saletti D, Pattofatto S, Bousson V, Laporte S: Links between mechanical behavior of cancellous bone and its microstructural properties under dynamic loading. J Biomech 48:498, 2015
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Figure Legends: Figure 1. Micro-CT reconstructed images showing trabecular microstructure of the group SHAM analyzed.
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Figure 2. Micro-CT reconstructed images showing trabecular microstructure of the groups DR analyzed. Figure 3. Micro-CT reconstructed images showing trabecular microstructure of the groups DCR analyzed.
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Figure 4. Significant differences observed in the study for bone volume fraction (BV/TV). *Significant differences (p<0.05).
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Figure 5. Significant differences observed in the study for Fractal Dimension (FD). *Significant differences (p<0.05). Figure 6. Significant differences observed in the study for Total Porosity. *Significant differences (p<0.05).
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Figure 7. Significant differences observed in the study for Structure Model Index (SMI). *Significant differences (p<0.05).
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S0278-2391(17)31544-6
DOI:
10.1016/j.joms.2017.12.019
Reference:
YJOMS 58101
To appear in:
Journal of Oral and Maxillofacial Surgery
Received Date: 12 September 2017 Revised Date:
20 December 2017
Accepted Date: 20 December 2017
Please cite this article as: Abdala-Júnior R, Gonzalez Cortes AR, Aoki EM, Ferreira S, Cerqueira Luz JG, Arita ES, Xavier de Oliveira J, Impact of temporomandibular joint discectomy on condyle morphology: an animal study, Journal of Oral and Maxillofacial Surgery (2018), doi: 10.1016/ j.joms.2017.12.019. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Impact of temporomandibular joint discectomy on condyle morphology: an animal study Reinaldo Abdala-Júnior, DDS, MS (Graduate student)1
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Arthur Rodriguez Gonzalez Cortes, DDS, PhD (Postgraduate fellow)1,2,3 Eduardo Massaharu Aoki, DDS , MS (Graduate student)1 Simone Ferreira, PhD (Researcher)4
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João Gualberto Cerqueira Luz, DDStabe, PhD (Associate professor)4 Emiko Saito Arita, DDS, PhD (Associate professor)1
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Jefferson Xavier de Oliveira, DDS, PhD (Associate professor)1
1
Oral Radiology Division, School of Dentistry, University of Sao Paulo, Brazil Martinos Center for Biomedical Imaging, Department of Radiology, Massachusetts General Hospital, Charlestown, MA 02129, USA 3 Harvard Medical School, Boston, MA 02115, USA 4 Department of Oral Surgery, School of Dentistry, University of Sao Paulo, Brazil
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2
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Corresponding author: Dr. Reinaldo Abdala Júnior , Department of Stomatology, School of Dentistry, University of São Paulo, Av. Lineu Prestes, 2227. Zip code: 05508-000. São Paulo, SP, Brazil. Telephone and fax number: +55 11 3091-7831. E-mail address: [email protected].
Running Title: Impact of discectomy on condyle morphology
ACCEPTED MANUSCRIPT
Abstract
Purpose: Temporomandibular disorders (TMD) lead to parafunctional activity
RI PT
that may alter bone remodeling of mandibular components. This animal study aimed to assess the impact of temporomandibular joint discectomy on condyle bone microarchitecture.
SC
Material and Methods: A total of 30 one-month-old Wistar rats were assessed and divided in three equal groups (two tests and one control) of 10. The first test
M AN U
group underwent disc removal (DRm; the second test group underwent disc and condylar cartilage removal (DCR); and the 10 remaining rats were analyzed as sham-operated controls (CTR), following a split-mouth design. Rats were sacrificed two months after surgery, and the respective mandibles were scanned
TE D
with micro-CT for quantitative morphometric analysis.
Results: There were significant differences among the three groups analyzed (DR, DCR and CTR) for Bone volume fraction (BV/TV, p=0.044), Structure model
EP
index (p<0.001), Fractal Dimension (p=0.024) and porosity (p=0.023). In addition, operated and contralateral non-operated sides significantly differed for all
AC C
variables in at least one of the test groups (p<0.05), but not in the control group (p>0.05).
Conclusion: Within the limitations of this study, the present results suggest that discectomy may lead to alterations of the mandibular condylar morphology.
ACCEPTED MANUSCRIPT
Keywords: Temporomandibular joint; temporomandibular disorders; micro-
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computed tomography, alveolar bone; Mandibular condyle.
Introduction
It is known that parafunctional activity may lead to morphological changes
SC
of maxillofacial hard and soft tissues.[1-3] Such conditions can be triggered by alterations of temporomandibular joint (TMJ) components, which may have a
M AN U
significant impact on mandibular structures such as the condyle.[1] Previous studies observed that TMJ disk integrity contributes to the healing process in cases of condyle fracture.[4, 5] Temporomandibular
disorders
(TMD)
involve
alterations
of
joint
TE D
components that can be treated with several methods, such as physical therapy, occlusal adjustment, medications or surgeries.[6] One of the surgical modalities that has been used to treat painful TMD cases not responding to non-surgical
EP
treatment is the discectomy. Although this is a predictable treatment modality, it may lead to postoperative clinical conditions such as condyle flattening and
AC C
sclerosis.[7, 8]
Despite of the above-mentioned evidences, little is known on the
microstructural effects of discectomy on condyle bone morphology. In this context, micro-computed tomography (µCT) has been used to detect alterations of micro-architecture of rat trabeculae in images with adequate isotropic resolution. [9]
ACCEPTED MANUSCRIPT
Thus, the aim of this study was to assess the impact of TMJ discectomy
RI PT
on the condyle bone microarchitecture of rats.
M AN U
Study Sample
SC
Material and Methods
A total of 30 one-month-old male Wistar rats (average weight: 70.1±3.6 grams) were included in this study. All rats received a diet based on granulated commercial rodent (Labina, Agribrands-Purina Ltda, Paulinia, Brazil) and water
TE D
ad libitum. The handling of animals was in accordance with the ethical principles proposed by the Brazilian College of Animal Experimentation. Approval was obtained from the animal ethics, care and use committee of the university of this
EP
study (protocol number: 035-2015).
AC C
Groups’ definition
Rats were classified into three equal groups (disc removal (DR); disc and
condylar cartilage removal (DCR); and sham-operated controls (CTR)), following a split-mouth design:
In the DR group: rats undergoing disc removal surgery in one side, and no surgery in the contralateral side (n=10); in the DCR group: Rats undergoing both
ACCEPTED MANUSCRIPT
disc and condylar cartilage removal in one side, and no surgery in the contralateral side (n=10); in the CTR group: Rats that underwent sham surgery in
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one side, and no surgery in the contralateral side (n=10).
Surgical procedures
All procedures were performed under general anesthesia induced by
SC
xylazine hydrochloride 10 mg/kg (Rompum, Bayer, Porto Alegre, RS, Brazil) and ketamine hydrochloride 25 mg/kg (Dopalen, Vetbrands, Paulínia, SP, Brazil).
M AN U
The present study followed a previously described methodology for conducting split-mouth design studies on local interventions in the TMJ.[5, 10-12] Only the right side of all rats underwent surgical interventions while the left side remained intact. Surgical flaps were opened with a one-centimeter-long
TE D
horizontal incision that followed the extension of the zygomatic arch, following its identification by palpation.
Each group underwent only one surgical intervention, described as
EP
following: in the CTR group, flaps were secured with no actual intervention on the TMJ (sham-operated). In the DR group, the articular disc was removed using
AC C
scalpel and straight forceps. Finally, in the DCR group, rats underwent both articular disc and condylar cartilage removal. The latter was carefully performed using a specific scalpel blade (# 15). Following the above-mentioned procedures, flaps were secured by layers
with monofilament sutures in all groups. After a two-month postoperative follow-
ACCEPTED MANUSCRIPT
up, all rats were sacrificed by lethal dose of anesthesia. Then, the heads were
RI PT
removed, fixed in formalin and dissected.
Micro-CT
SC
Multiplanar and volumetric reconstructions of the condyle bone of all specimens were obtained using a Micro-CT system - SkyScan Model 1176
M AN U
(Skyscan; Kontich, Belgium) with the following imaging protocol: 50 kVp, 500 mA, 16 bits, 8.71µm of resolution, 0.5 degree step of the rotation angle and 360 degrees of rotation. The specimens were mounted on a low-density foam base and fixed by a PVC film (Polyvinyl Chloride) before the scan. Projections and
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volumetric images were reconstructed using NRecon software (Bruker, Belgium). A volume of interest [13] enclosing the entire condyle area was delineated and considered in all analyses of trabecular bone micro-architecture. To ensure
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uniformity of the VOI selection, the hemimandibles had been previously aligned on the X, Y and Z in the Data Viewer software (Bruker, Belgium). Then, 2D and
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3D morphometric analysis were performed using Skyscan CT-Analyser software (Bruker, Belgium).
The following parameters of trabecular microstructure were quantitatively
assessed: bone volume fraction (BV/TV), which is the ratio of bone volume to the total volume of the sample, and has been considered a measure of strength and stiffness of cancellous bone;[14] trabecular separation (Tb.Sp), which is also
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related to bone strength and is usually altered with age;[15] fractal dimension (FD), which is a statistical texture analysis associated with bone stiffness;[16] connectivity density (Conn.Dn), which is a predictor of vascular network;[17] total
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porosity; and structure model index (SMI), which normally varies between 0, for perfect plates, and 3, for perfect rods. Negative SMI values are associated with
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isolated marrow spaces.
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Statistical analysis
The sample size used was determined considering an effect size of f = 0.6, to give the study a power of 80% (type II β error = 0.2) at a significance level of 5% (type I α error = 0.05), resulting in a total sample size of 30 rats, equally
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distributed into three groups. Normality was calculated for all outcome variables (BV/TV, Tb.Sp, FD, Conn.Dn, porosity and SMI) with the Shapiro-Wilk test. Paired Student's t-test was used to compare the variables between left and right
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sides of the same mandibles (i.e within-subject measurements). In addition, results for the right (operated) side of all groups (DR, DCR and CTR) were
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compared (i.e. between-subject measurements) with analysis of variance test (One-way ANOVA). Post-hoc analysis was conducted with Tukey HSD. Because of our split-mouth study design, the aforementioned analyses did not need to be adjusted to confusion variables such as age and gender. A 5% significance level for all parameters (p <0.05) was adopted. The software used for statistical analysis was SPSS (IBM SPSS Version 20.0 Chicago, IL, USA).
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Results
Normality was confirmed for all variables (p>0.05). Regarding within-
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subject measurements, operated (Figures 1, 2 and 3) and contralateral nonoperated sides significantly differed for all significant variables in at least one of the test groups. For BV/TV (Figure 4), both DR and DCR differed between sides
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(p=0.031 and p=0.019, respectively). For FD (Figure 5), total porosity (Figure 6), and SMI (Figure 7), only the DCR group showed significant differences (p<0.001,
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p=0.003, and p=0.001, respectively). No significant differences between sides were observed in the control group (p>0.05).
Furthermore, there were significant between-subject differences among the three groups analyzed (DR, DCR and CTR) for BV/TV (p=0.044), SMI (p<0.001), FD (p=0.024) and porosity (p=0.023). All aforementioned differences
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were confirmed by Tukey’s Post-hoc test (p<0.05). No other variables differed
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Discussion
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significantly among groups (p>0.05).
Discectomy leads to morphologic alterations of the mandibular condyle,
which may have an impact on occlusal function. In order to adapt the bone to the new mechanical demands, increased mechanical loads will lead to new bone formation and remodeling. Similarly, a lack of mechanical loads might lead to low bone trabecular and mineral density.[18, 19]
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In the present study, discectomy and subsequent cartilage removal led to worsen morphometric conditions of the condyle. TMJ stability depends on a muscular condition that is achieved when the condyles are located in their most
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superoanterior position regarding the articular fossae, with the disc joint itself interposed between the aforementioned two structures.[20] In addition, TMD has been found to alter jaw muscles properties, such as functional efficiency, force of
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contraction and electrical activity, leading to lack of stimulation on the condyle bone.[21] Our hypothesis that the DCR group would present worsen
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morphometric conditions than both DR and CTR groups was confirmed, supporting previously described evidences.[22] In addition, DCR groups presented significant within-subject differences in more morphometric variables than DR and CTR groups. The latter, in particular, did not present any significant
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differences between operated and non-operated sides. Such findings are in agreement with a previous similar study in the literature.[12] On the other hand, there was no significant difference in some of the
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morphometric parameters, such as trabecular separation, among the three groups. This could indicate the occurrence of a compensatory mechanism of
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bone morphometry triggered by the lack of condylar cartilage due to the great adaptive capacity of immature TMJ.[5, 23] One limitation of this study, however, is that no direct assessments of
muscular activities could be performed for the groups analyzed. Future clinical and animal studies monitoring of the activity of jaw muscles would be recommended to understand compensatory growth mechanisms involving the
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TMJ, taking into consideration that mandibular growth is directly affected by muscular activity.[3, 19, 24] Little is known on the effects of TMJ alterations on mandibular bone sites.
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To our knowledge, this is the first study addressing the effect of discectomy on the condyle bone morphometry, offering microscopic evidences that support studies that found clinical alterations in cases of discectomy, such as condyle
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flattening and sclerosis.[7, 8] Micro-CT has been considered the best option to quantifying 3D bone microarchitecture, allowing for obtaining direct volumetric
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and morphometric measurements of trabecular bone features, contrasting with 2D histological assessment based on stereological methods.[9, 25, 26] Furthermore, micro-CT is the most used method to assess bone morphometric variables. Laboratorial assessments based on finite element analyses concluded
properties.[27-29]
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that trabecular microstructure has a significant impact on bone biomechanical
In conclusion, within the limitations of this study, the present results
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suggest that either removal of the articular disk or concomitant removal of both disc and articular cartilage may lead to worsen condyle bone morphometric
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conditions, as compared to the contralateral non-operated sides.
Acknowledgements: The authors declare that there are no conflicts of interest in this study and that they agree and have viewed this submission. Two master scholarships were granted to RAJ and EMA by the Coordination for the Improvement of Higher Education Personnel (CAPES – Brazil). A post-doctoral scholarship was granted to ARGC by the National Council for Scientific and Technological Development (CNPq)—Science without Borders, Brazil, no. 232643/2013-0.
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Figure Legends: Figure 1. Micro-CT reconstructed images showing trabecular microstructure of the group SHAM analyzed.
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Figure 2. Micro-CT reconstructed images showing trabecular microstructure of the groups DR analyzed. Figure 3. Micro-CT reconstructed images showing trabecular microstructure of the groups DCR analyzed.
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Figure 4. Significant differences observed in the study for bone volume fraction (BV/TV). *Significant differences (p<0.05).
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Figure 5. Significant differences observed in the study for Fractal Dimension (FD). *Significant differences (p<0.05). Figure 6. Significant differences observed in the study for Total Porosity. *Significant differences (p<0.05).
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Figure 7. Significant differences observed in the study for Structure Model Index (SMI). *Significant differences (p<0.05).
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