This study included eleven patients with temporomandibular joint disorders, three of which had neurological conditions. A UCLA IRB was generated and accepted (IRB approved number 13-000027) to receive and process samples at UCLA.
We established a collaboration with the Community Clinic Association of Los Angeles County (CCALAC). Potential TMD patients were identified based on their symptoms at CCALAC associated (member) clinics. They were referred to the dental practice of Dr. Gary Demerjian, who is part of the evidence-based decision practice-based research network (EBD-PBRN) associated with our lab at UCLA School of Dentistry. Patients signed a consent form at the clinic of Dr. Demerjian allowing clinical evaluation of their TMJ joint, and collection of their left and right TMJ synovial fluid as well as whole saliva samples.
Clinical Examination was performed by Dr. Gary Demerjian whose practice is limited to TMJ and Dental Sleep Medicine. Diagnosis was based on clinical examination including a review of the patient’s medical history, clinical symptoms, palpations of the head and neck, intra-oral exam, joint vibration analysis, electromyography, CBCT with an iCAT imaging system, analyzed with Vision software. iCAT is a high frequency, constant potential, fixed anode X-ray source (120 kVp, 3–8 mA). It incorporates a flat panel, amorphous silicon image detector at 20 cm × 25 cm, with a cesium iodide conversion layer. Scan dimensions are 17 cm × 13 cm. BioPak system was used for surface EMG measurements and JVA of the TMJ. Surface EMG pads were used on the bilateral temporalis, masseters, anterior digastric, and sternocleidomastoid. JVA (Electrovibrography) was used to measure and document the disc displacement during the examination. Treatment included fabrication of patient tailored oral orthotic and periodic adjustments.
Left and right TMJ synovial fluid was obtained as part of the lavage of the joint compartments. A 27 gauge (27G × 1/4 inch − 0.4 × 32 mm) syringe with a hypodermic needle was used to aspirate the TMJ. A 2 ml of 2% LIDOCAINE (20 mg/ml) solution was used in the lavage protocol and the amount of samples obtained per joint was in the range of 250–1000 µl (mixture of 2% LIDOCAINE and SF). Patients were also asked to produce fresh whole saliva samples. All samples were collected in sterile tubes containing sterile buffered solution of chelating agents (1 mM EDTA). They were coded and stored frozen until transported on dry ice to UCLA to be stored at − 80 °C until processed. Samples were batch assayed for IL-1β (Qiagen USA; linear limit of detection 17.9 pg/ml), TNF-α (Qiagen USA; linear limit of detection of 21.4 pg/ml), IL-6 (Qiagen USA; linear limit of detection of 14.0 pg/ml), IL-17A (Qiagen USA; linear limit of detection of 28.4 pg/ml), SP (Cayman Chemicals; linear limit of detection of 8.2 pg/ml), CGRP (Cayman Chemicals; linear limit of detection of less than 5 pg/ml) and PAD-4 (Cayman Chemicals; a range of 1–1000 U/ml and LLOQ 31.3 U/ml) with commercially available ELISA kits.
Clinical observation data were correlated with the concentration of the biomarkers (or parameters) when appropriate using Spearman’s rho correlation coefficient. Patients were ranked based on clinical severity of TMD. Comparisons of left and right TMJ (synovial fluids) were performed by Matched-pair t test (or rank-sum when non-parametric). The comparison of left and right synovial fluid and whole saliva within each subject was performed using one-way ANOVA followed by Scheffe Post Hoc Comparison with Bonferroni Correction (or when non-parametric kruskal–Wallis and Wilcoxon). Level of significance was set at α = 0.05 for all statistical analyses.
We also collaborated with Medical Imaging Center of Southern California for fMRI and neuroimaging studies. The fMRI study for patients 9–11 was performed using a Siemens Magnetom Verio 3 Tesla MRI clinical open system scanner (Siemens Healthcare GMbH, Erlangen, Germany) and the following sequences were used: (1) MPRAGE. (2) Resting state network. EPI sequences for this resting state BOLD scan was post processed on a 3D workstation. ICA was performed separating out the various networks. (3) ASL. (4) Tractography and FA. In this study, a quantitative volumetric analysis was not done. Images were visually inspected for gross anatomical abnormalities by experienced observers (neuroradiologists), blind to participant identities without using a quantitative regional analysis and normal database. When we refer to morphology of brain in this study, it is the gross anatomic evaluation of the brain qualitatively as oppose to quantitatively.
Diffusion Weighted Imaging (DWI), can be post-processed to create Diffusion Tensor Imaging (DTI) white matter tractography maps. From the diffusion tensor, diffusion anisotropy measures such as, the fractal anisotropy (FA) can be computed, a scalar value (between zero and one) representing an indirect assessment of the myelination integrity of white matter tracts in the brain. A value closer to zero means diffusion is isotropic, unrestricted (or equally restricted) showing loss of tissue organization, the myelin sheath. The corpus callosum, which connects the two cerebral hemispheres, is the largest white matter fiber bundle in the human brain, mostly traveling in a straight line. This is important because whenever there is a curve in white matter an artificial drop in FA occurs. Corpus callosum has been focus of many studies due to its structural characteristics. Many studies have focused on detecting the morphological changes in white matter integrity that occur in various disorders, particularly in Corpus Callosum, which serves as a sample of brain condition [54, 55]. Five regions of corpus callosum: genu, anterior body, mid body, posterior body and splenium were chosen as region of interest (ROI) in this study and were compared with the average value of the same ROI’s obtained from a normal database. The normal database was meticulously complied from 62 healthy controls ranging in age from 17 to 65, all right handed, at the Medical Imaging Center of Southern California using a Siemens Magnetom Verio 3 T MRI clinical open system scanner (Siemens Healthcare GMbH, Erlangen, Germany) excluding subjects with any brain abnormalities, neurological pathology, drug addiction, hypertension and diabetes detected by MRI scan. In order to minimize variations, the MRI scanner and the techniques were standardized by using the same scanner and post processing techniques creating the normal database, and the individual patient analyses. The post-processing for DWI was done on a Philips DynaSuite Neuro offered by Invivo post-processing workstation, which allows combining anatomical images and results, such as DTI or perfusion color maps, fiber tracks, and fMRI activations in a single 2D and 3D display. Diffusion weighted images were loaded on to Invivo workstation. They were first processed by performing a skull mapping, then the images were fused with MPRAGE in order to create tractography (anatomic map of tracts), then color mapping was done using DynaSuite workstation. The software is able to depict white matter bundles by direction. Left to right by this coding is blue, up and down is red, and anterior to posterior is green. ROI’s are taken by manual visual placement in axial plane at midline. The imaging parameters were as follows: Repetition time (TR) = 8400, eco time (TE) = 104, slice thickness = 3.5 mm, b = 0 and 1000 (images are automatically diversified to 1000).
Arterial Spin Labeling (ASL) generates an image by magnetically labeling water molecules as an endogenous tracer as they travel in blood. Selective radiofrequency (RF) irradiation inverts the magnetization of arterial blood water in the region or plane to which it is applied, usually in the neck for brain perfusion, and a downstream measurement is taken as labeled spins exchange in brain. In most ASL methods, the resulting images are compared to control images where inversion pulse is not applied. The difference reveals the perfusion, indirectly related to the quantification of cerebral blood flow (CBF) in units of milliliters of blood per 100 g of tissue per minute [56]. In this study we used a Siemens protocol offered on the Magnetom plus workstation. The 2D acquisition was processed using syngo image processing software offered by Siemens Healthineers. The heat map technique was used where flow is depicted in color-coding from red to dark blue. Red means more flow dark blue means less flow. We window it such that thalami are viewed, or identified bilaterally as red, visualizing blood flow to the brain regions.
Patients 1–8 had clinical examinations; synovial joint fluid and saliva samples were analyzed for correlation. Patients 9–11, who had TMD and comorbid neurological conditions, were clinically evaluated and participated in fMRI studies. A detailed medical history and diagnostic examination report is provided below.
Patient 9: reflex sympathetic dystrophy (RSD)/complex regional pain syndrome (CRPS)
Subject
A 40-year-old female presented with reflex sympathetic dystrophy and dystonia. Her reported symptoms were facial pain, jaw pain, migraine headaches, eye pain, dizziness, fatigue, muscle twitching, neck pain, ringing in the ears, pain when chewing and visual disturbances (limited field of vision).
Her medical history consists of neuralgia, autoimmune disorder, low blood pressure, muscle spasms, injury to the teeth, mouth, face and neck, kidney problems, muscle aches, hypoglycemia, muscle shaking, prior orthodontic treatment, poor circulation, intestinal disorder, bruising easily, chronic fatigue, depression, insomnia, psychiatric care, painful joints, tired muscles and difficulty concentrating.
She indicated moderate bilateral frontal and parietal headaches and severe bilateral temporal headaches lasting for hours, bilateral jaw pain on opening and chewing, jaw pain on the right while at rest, jaw clicking, teeth clenching, pressure behind the eyes, eye pain, blurred and double vision, photophobia, tinnitus, ear pain, difficulty swallowing, limited opening, neck pain, shoulder pain, thyroid enlargement burning tongue and dry mouth.
Clinical examination
Moderate pain was elicited upon palpation of left middle and posterior temporalis, and right anterior digastric. Severe pain was elicited upon palpation of bilateral temporalis, right middle and posterior temporalis, bilateral TMJ capsule, bilateral poster joint, bilateral masseter, bilateral posterior digastric, bilateral sternocleidomastoid, bilateral anterior, middle and posterior scalene, bilateral splenius capitus, bilateral trapezius. Provocation test elicited bilateral joint pain. Clinical examination revealed left TMJ click on opening and closing. Mandibular range of motion was 44 mm, maximum protrusion 10 mm, left and right excursions of 8 mm. Skeletal morphology and posture screening revealed facial asymmetry and a higher left shoulder. Intra-oral examination revealed maxillary and mandibular tori. She presented with a class I molar relationship, 4 mm overbite and 3 mm overjet in centric occlusion. Imaging records consisted of a maxillofacial CBCT and fMRI. Imaging examination revealed bilateral flattening on the anterior surface of the condyles, left condylar pitting/cratering, right bifid condyle, left posterior and superior displaced condyle.
Patient 10: Cervical dystonia
Subject
A 32-year-old female presented with pronounced cervical dystonia. Her symptoms were: neck pain, shoulder pain, back pain, fatigue, migraine headaches, headaches, muscle twitching, dizziness, throat pain, sinus congestion and visual disturbances. She suffers from moderate bilateral temporal, occipital and parietal daily headaches lasting for weeks, upper, middle and lower back pain, limited movement of her neck, shoulder pain and stiffness, pain behind the ears, and tingling in the hands. She has sensitivity to light, sound and touch to her head. She receives Botox injections into the neck and shoulder muscles every 3 months. She reports numerous head and spinal injuries throughout her life.
The patient has been diagnosed with Cervical Dystonia, fibromyalgia, Bell’s palsy, and C2–3 radiculopathy. Past history of surgeries are removal of third molars and gall bladder. The patient further reports a history of low blood sugar, muscle spasms, muscle aches and shaking, sinus problems, chronic fatigue, cold hands and feet, depression, dizziness and difficulty concentrating. List of medications taken are Docusate sodium, Fentanyl Transdermal System, Gabapentin, Oxycodone, Savella, Tizanidine HCL, Norgestimate-Ethinyl Estradiol Tablets, and Lunesta.
Clinical examination
Dental examination revealed a left TMJ click on opening. Mandibular range of motion measurements revealed maximum inter-incisal opening of 40 mm, maximum protrusive movement of 8 mm, lateral excursive movements of 7 mm right and 5 mm left. Posture screening revealed facial asymmetry, forward head posture and a higher right shoulder in relation to the left. Intraoral examination of the teeth revealed a molar class I relationship with 4 mm overjet and 2 mm overbite. Wear facets were noted on the dentition and missing third molars. Severe tenderness was elicited upon palpation of bilateral temporalis, masseters, lateral capsule, posterior joint space, sternocleidomastoid, anterior digastric, anterior scalene, middle scalene, posterior scalene, occipital, splenius capitus, trapezius.
Diagnostic tests included electromyography, photography, videography and thermography documentation. EMG’s were placed on her bilateral temporalis, masseter, anterior digastric and Sternocliedomastoid muscles, while the patient was seated in a resting position. Right SCM was contracting while in the resting position and upon delivery of the orthotic, her muscle activity normalized on the EMG’s. Photographs were taken demonstrating her cervical dystonia. Video graphic documentation demonstrated her history and progress of care. Thermographic images showed an increase in surface temperature of the head, neck and upper back. CBCT scan of the maxillofacial examination revealed posterior displacement of both condyles, flattening of the posterior portion of both condyles, and pharyngeal airway impingement. MRI study indicated bilateral articular discs displaced in the closed mouth position, with reduction in the open mouth position.
Patient 11: Sympathetic dystrophy and chronic pain
Subject
36-year-old male presented with joint pain in his hands and feet for over 25 years. He complains of daily back pain, neck pain, shoulder pain, throat pain, fatigue, eye pain, jaw pain, facial pain, numbness of his chin and pain when chewing. His frequent symptoms are ear congestion, ear pain and headaches. His history consists of four rear end collisions. He started having migraine headaches 2 years after a roller-skating accident, where he broke his arm during his middle school years. He reports being in pain, his whole life. He has been diagnosed with sympathetic dystrophy, irritable bowel syndrome, acid reflux and paradoxical sphincter contractions.
Clinical examination
Clinical examinations revealed sever pain upon palpation of bilateral temporalis, lateral TMJ capsule, posterior TMJ, masseters, anterior digastric, sternocleidomastoid, trapezius, occipital and splenius capitus. Intra-oral examination revealed maxillary and mandibular tori, buccal exostosis, scalloped lateral borders of the tongue, wear facets on the dentition and gingival recessions. Maximum inter incisal opening of 40 mm, right lateral excursion of 10 mm, left lateral excursion of 12 mm and protrusion of 11 mm. Molar relationship of class I on the right and class II on the left, overbite of 4 mm, overjet of 4 mm and the mandibular midline being to the left by 1 mm in relation to the maxillary midline. CT scans of the maxillofacial examination revealed right condylar flattening on the anterior surface and bilateral posterior and superior displacement of condyles in relation to the temporal fossa.