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Morphofunctional analysis of temporomandibular joint after bilateral discectomy and discopexy : preclinical study
Publication . Ângelo, David; Salvado, Francisco, 1957-; Gil, Florencio Monje; González-Garcia, Raúl
INTRODUCTION The temporomandibular joint (TMJ) is the most used joint in the human body, with over 2000 movements per day, being essential for everyday functions (e.g. mastication, speech, deglution and yawning). The TMJ is responsible for the relation between the mandibular condyle and glenoid fossa of temporal bone. This joint contains an articular disc, an important functional unit interposed between the bony structures, contributing for a congruent movement of this joint. TMJ disorders (TMD) have a prevalence of 34% and represent the main non-tooth origin cause of orofacial pain, leading to high morbidity in severe cases (category 3-5 Dimitroulis classification). In those cases, the treatment is mostly surgical, being the common surgical options discopexy or discectomy. The outcomes of these interventions are a topic of debate, namely in discectomy, once TMJ is left without the TMJ disc. Moreover, in the available scientific literature it is not possible to state any randomized study, blind and with control group, both in human or animals, evaluating the effect of those interventions in TMJ. OBJECTIVES The main goal of this dissertation was evaluating the effect of TMJ discectomy and discopexy in animal model (Black Merino sheep), examining their impact in histologic, imaging and kinematic outcomes. Specific goals: 1. Characterisation of the adequate animal model for preclinical studies in the TMJ. 2. Characterize the anatomy and biomechanics of the native TMJ disc of Black Merino sheep. 3. Develop a study design for preclinical investigation in the TMJ, according to the ARRIVE guidelines 4. Application of the proposed study design on the Black Merino animal model. 5. Propose and test pilot outcomes on the kinematic mastication for preclinical studies in the TMJ and evaluating their impact on bilateral discectomy and bilateral discopexy in Black Merino sheep. 6. Evaluating the histologic and imaging impact of bilateral discectomy and bilateral discopexy in Black Merino sheep. METHODS To achieve the define goals, the following activities were developed and proposed: 1) Characterisation of the adequate animal model for preclinical studies in the TMJ: 15 female Black Merino sheep heads of healthy animals with 40-50 Kg were used to describe the surgical, topographic and histologic anatomy of the TMJ, comparing with the human joint. In a second phase, histologic and biomechanical analysis of the disc were performed. For the anatomical characterisation, a surgical dissection was performed exposing and identifying the TMJ structures. To analyse the TMJ disc, a microsurgical dissection was performed and the TMJ discs were submersed in an impregnated solution (Colorbond), allowing a 3D scanning preserving the morphology of the native disc. To the histologic analysis, the TMJ was removed in block, decalcified in formic acid, included in paraffin and stained with hematoxilin & eosin and orcein. For the biomechanical evaluation, 9 discs were randomized (3 were tested to compression, 3 to anteroposterior strain and 3 to mesiolateral strain). 2) Characterize the anatomy and biomechanics of the native TMJ disc of Black Merino sheep: a microsurgical extraction of 6 discs was completed, removing all the muscular attachments. The discs were submersed in an impregnate solution to preserve their native morphology. A white light system scanner was used, replicating a 3D virtual model with high precision. 3) Develop a study design for preclinical investigation in the TMJ, according to the ARRIVE guidelines: a randomized preclinical trial with 10 sheep was proposed with allocation in 3 intervention groups: bilateral discectomy, bilateral discopexy, sham surgery and one reserve sheep was allocated in a reserve group. A baseline pre-intervention record of body mass, mastication time for 150gr of pellets and rumination kinetics was included. In addition, imaging by computed tomography (CT) was suggested before surgical intervention. It was proposed that the animals would be submitted to surgical intervention and evaluated every 6 months, being posteriorly sacrificed. After sacrifice, a new CT should be done for imaging scoring and discs ought to be prepared for the histologic evaluation. 4) Application of the proposed study design on the Black Merino animal model: the study started in December 2015, the baseline records occurred in January 2016, and the surgical interventions took place in February 2016. According to the established protocol, the animals were monitored for 6 months (with various assessments being performed in specific time points) and were sacrificed in August 2016. 5) Propose and test pilot outcomes on the kinematic mastication for preclinical studies in the TMJ and evaluating their impact on bilateral discectomy and bilateral discopexy in Black Merino sheep: nine specific cages were constructed with a frontal window where all the animals were placed at 08:30 am. The time to eat 150gr of dry pellets Rico Gado A3® was assessed by a chronometer. Following, the animals were placed in their natural habitat and around 01:00 pm they returned to the boxes to record the kinetic rumination of 15 cycles. With adequate software, the trajectory of the jaw was designed during rumination and the time for each cycle was calculated, as well as the ruminant area. Next, the body mass was obtained in a certified balance. 6) Evaluating the histologic and imaging impact of bilateral discectomy and bilateral discopexy in Black Merino sheep: after sacrifice, a cranium CT was performed and the TMJ block was removed. Each block was prepared for histology. In occultation, the different evaluators scored the joint using appropriate scales. 1) The obtained surgical anatomy was similar to the human, with a direct access to TMJ through a pre-auricular incision. The size of the anatomic structures (e.g. temporal fossa, mandibular condyle, inter-articular space and TMJ disc) were also similar to the human. In addition, high similarity was also obtained in the disc histology and biomechanics. A slight concave mandibular condyle was noticed, which is convex in humans. 2) With the proposed method, it was possible to accurately replicate the morphology of the native TMJ disc of Black Merino sheep. The average size was 20.93±1.33mm in the mesiolateral orientation and 11.2±0.78mm in anteroposterior orientation. The thickness of the anterior band was 1.03±0.06mm, the central zone 0.76±0.08mm and the posterior band 1.23±0.07mm. The morphology of the fresh disc showed a slight convexity of the inferior surface, consistent to an adaptive outline to the mandibular condyle. 3) The randomized and blinded study design, with allocation in 3 different bilateral interventions, respecting the ARRIVE guidelines was executed with accuracy and proved to be feasible in Black Merino sheep. The video caption of 15 rumination cycles was achievable, showing this animal the ability to stand still during the rumination. 4) Various histologic changes were noticed in the discectomy and discopexy groups. As expected, the control group presented the main characteristics of the normal TMJ. In the discectomy group, fibrillation and loss of typical laminar structure, with an increased population of proteoglycan stain in all layers and vascular invasion, were observed. In some cases, osteochondral changes were also noticed. The blinded scoring showed statistical significance for the discectomy group only. While no statistical significance, with no loss of the laminar surface, was detected in the discopexy group, a marked proteoglycan increase was noticed. 5) The blinded evaluation of CT images showed statistical significance only for the discectomy group (R2 corresponding to 92.9% of degeneration for global appreciation). In the discopexy group, slight differences were noticed without reaching statistical significance. 6) For both surgical techniques, significant changes were noticed for mastication time in the first month post-operative. Those changes gradually returned to normal and 6 months after surgery no changes were observed. No rumination movements were detected in T1 a T2 after surgery in the discectomy group. In rumination evaluation, a significant difference was stated in T5 for discopexy group with an increased time per cycle. No other changes occurred, neither for rumination time or area. 7) In the first month after surgery, discectomy and discopexy groups loss body mass. However, with the progress of the study, those animals were capable to increase body mass; discectomy group was only capable to return to original weight, and discopexy groups could follow the control group increasing 8% and 8.2% respectively. No significant changes were noticed in the different groups. CONCLUSIONS The Black Merino sheep has proven to be an adequate animal model to conduct TMJ preclinical trials, considering the surgical anatomy, biomechanics and histology like human. Because it is a ruminant animal, it may have an increased value in further studies, examining interposal biomaterials or TMJ prosthesis, and allowing to accurately test the material stress. The proposed randomized, blinded, with bilateral interventions study was feasible and reproducible for TMJ investigation in Black Merino sheep. Discectomy promoted a severe degenerative process in histologic and radiologic analysis, with a repercussion in mastication (only in the first month post-surgery). Discopexy did not induce significant degenerative changes in the TMJ. With a significant role in tissue regeneration, bioengineering and regenerative medicine will be critical to increase and optimize the current surgical techniques, mostly discectomy.
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Fundação para a Ciência e a Tecnologia
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5876
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UID/Multi/04044/2013