Salivary biomarkers in chronic muscle pain

Abstract

Background: Muscle related temporomandibular disorders (TMD myalgia), one of the most common orofacial pain conditions, is characterized by facial pain and often accompanied by jaw movement limitations. Although the underlying biological mechanisms are still unclear, a cluster of proteins and peptides is assumed may mirror the pathophysiology. These proteins and peptides may be measured in a simple non-invasive saliva sample. However, the variability in saliva sample collections and analyses should be kept to a minimum to ensure that reproducibility testing can accurately assess changes between health and disease state.

Aims: This thesis investigated whether saliva can be used to sample algogenic substances that can serve as molecular biomarkers for TMD myalgia. The specific aims of the methodological section were to compare saliva collection methods and to evaluate the daily variation of pain-related mediators. The specific aims of the clinical section were to evaluate algesic mediators and the protein profile in saliva of TMD myalgia for potential diagnostic salivary biomarkers.

Material and methods: Saliva and blood samples were collected from healthy individuals (n=69) and patients diagnosed with TMD myalgia (n=39) according to the Diagnostic Criteria for TMD. Unstimulated and stimulated whole, parotid, and sublingual saliva were analysed. The protein profiles were investigated using two-dimensional gel electrophoresis followed by identification with liquid chromatography tandem mass spectrometry. Levels of nerve growth factor (NGF), calcitonin gene-related peptide (CGRP), and brain derived neuro-tropic factor (BDNF) were determined using western blotting based technology and multiplex electro-chemiluminescence assay panel. Glutamate, serotonin, and substance P (SP) were determined using commercially available methods.

Results: The results showed that different saliva collection approaches resulted in significant differences in the protein profile as well as in the expression of NGF, BDNF, CGRP, SP, and glutamate. Stimulated whole saliva showed least variability in protein concentration (35%) and was correlated to plasma levels of glutamate (rs = 0.56; P = 0.011). Unlike SP and glutamate, NGF and BDNF expressed a rhythmic variation in salivary expression with higher levels in the morning (P < 0.05). Patients with a diagnosis of TMD myalgia had significantly higher levels of salivary glutamate but lower salivary NGF and BDNF compared to controls (P < 0.05); in addition, the lower NGF and BDNF levels correlated to psychological dysfunction (rs > -0.462; P < 0.001). The quantitative proteomics data revealed 20 proteins that were significantly altered in patients compared to controls. The identified proteins are involved in metabolic processes, immune response, and stress response. Dissimilarities in protein profile and clinical variables were observed between TMD myalgia and myofascial pain.

Conclusion: The thesis highlights the importance of consistency in saliva collection app-roaches, including the timing of the collection. It displayed significant changes in pain specific mediators and protein profile in TMD myalgia and furthermore dissimilarities between subclasses indicating different pathophysiology. After extensive validation, potential salivary biomarkers can be combined with clinical features to better understand and diagnose TMD myalgia.