Molecular Reaction In Peripheral Blood Involving Slight Injury Of Rat Temporomandibular Joint And Masticatory Muscle Induced By Unilateral Anterior Crossbite

Occlusion has a close relationship with masticatory muscle and temporoamndibular joint(TMJ),while malocclusion may cause masticatory muscle and TMJ dysfunction.We recently reported that unilateral anterior crossbite(UAC)could induce obvious reactions in TMJ condyle cartilage and subchondral bone,including changes in molecular,histology and even gross morphology.TMJ consists of cartilage,bone and disc and so on,and has a close relationship with masticatory muscles.Accordingly,we are curious about how TMJ disc and masticatory muscles changes in UAC rats,and whether the changes in the TMJ and masticatory muscles can cause a reflection in the peripheral blood leukocyte.Then this article is aiming to the above two questions.Part I: Effects on rat temporomandibular joint discs induced by UACIn this part,54 female Sprague–Dawley rats were equally distributed into a UAC group and a sham-operated control group at 4,12,and 20 weeks(n = 9).And their TMJ discs were evaluated by gross and histomorphological observation and molecular biology assay of the markers related to the disc matrix.Results: No macro-or micro-morphological differences were observed between groups.However,there were degradative changes at the molecular level in the UAC group,showing a significant reduction in the mRNA and/or protein expression levels of many molecules with time(P < 0.05).The reduced molecules were as follows:(i)molecules related to the extracellular-matrix,such as Type-I collagen,decorin and fibromodulin;(ii)molecules related to chondrogenesis,such as Type-II collagen and aggrecan;and(iii)molecules related to osteogenesis,such as alkaline phosphatase and runt related transcription factor 2.The mRNA expression of vascular endothelial growth factor did not change.In contrast,fibronectin,which can promote wound healing,and its N-terminal fragment,which can induce cartilage degradation,were accumulated(P < 0.05).Conclusion: TMJ discs degradation was enhanced with time by the aberrant dental occlusion.Part II: Effects on rat masticatory muscle induced by UACIn this part,the alterations of the masseter,a representative jaw elevator,and the lateral pterygoid muscle(LPM),a representative jaw depressor,and their morphological and molecular biological reaction towards UAC cessation were examined.Results: Two weeks of UAC elicited mild injury of the two muscles.Myogenesis and protective reaction were detected as increases of ?B-crystallin expression in masseter from 3 days and in LPM from 2 weeks,and increases of desmin expression in both muscles at 2 weeks.A switch of fiber types from IIb to IIx existed in LPM,but not in masseter.Inflammatory responses shown by infiltration of inflammatory cells and increases in TNF-? mRNA expression appeared very mild in masseter from 3 days and in LPM only at 1 week.Increased mRNA expression of Type I and III collagen occurred in masseter from 3 days and in LPM from 1 week.All these responses were partially recovered by cessation of UAC.During the whole process,no obvious changes were noticed in the mitochondrial function indicated by the level of proliferator-activated receptor ? coactivator 1?,mitofusin-2 and voltage-dependent anion channel.Conclusions: UAC causes injury and very limited inflammatory and fibrosis adaption in masseter and LPM.Both muscles respond with myogenesis and protective while LPM,in addition,with muscle fiber isoforms alternation.Those alterations are partially recoverable by cessation of the dental stimulation at early stage.Part III:Molecular changes in rat peripheral blood leukocytes induced by long-term UACTo detect whether molecular biomarkers from the peripheral blood leukocytes(PBLs)engage in masticatory muscle and TMJ injury.The top enrichments of gene ontology category and gene fold changes in PBLs were detected by microarray analysis and compared between the UAC rats which had induced TMJ OA-like lesions and the age-matched controls(n=4).Another twenty-four rats were randomized to UAC and control groups at 12-and 20-week time points(n=6).The mRNA expression levels of the selected biomarkers derived from microarray analysis and their protein expression and location in the masseter,the lateral pterygoid muscle(LPM),alveolar bone,TMJ disc,mandibular condylar cartilage and subchondral bone,were detected using real-time PCR,western blot and immunohistochemistry assays.Results: Microarray analysis indicated three most involved genes,Fbxo32,Egr1,Ephx1 and Il10,in PBLs which were confirmed by real-time PCR detection.The protein expression levels of FBXO32 were increased in masseter after UAC operation,which indicating astrophy in masseter.The increased protein expression levels of the three detected molecules were demonstrated in cartilage and subchondral bone(P < 0.05),and that of EPHX1 in disc(P < 0.05),but none in alveolar bone.Immunohistochemistry revealed the increased distribution of EGR1,EXPH1 and IL10 positive cells predominantly in osteochondral interface,and EXPH1 additionally in TMJ discs.Conclusions: Our data indicate that the increased mRNA expression of Fbxo32,Egr1,Ephx1 and Il10 in PBLs could be taken as potential biomarkers for masseter astrophy and developed osteoarthritic lesions relating to osteochondral interface hardness changes induced by dental biomechanically stimulation.Part IV:Early gene marker in peripheral blood leukocytes of human and rats with TMJ OATo detect whether early growth response gene 1(EGR1)engages in TMJ OA lesions.Egr1 mRNA expression level of PBLs were detected in eight malocclusion patients without TMD signs and sixteen malocclusion patients having TMD signs with(eight cases)or without(eight cases)diagnosis of TMJ OA using cone beam computerized tomographic.Twelve rats were randomized to a control group and a UAC group induced with TMJ OA.The Egr1 mRNA expression levels of PBLs and its protein expression and location in the different orofacial tissue of each rat group was detected using real-time PCR,western blot and immunofluorescence assays.Results: Compared with malocclusion patients without TMD signs,the patients having TMD signs with/without TMJ OA diagnosis showed lower Egr1 mRNA expression level in PBLs.Such a difference was also revealed in UAC rats PBLs.The EGR1 protein expression level was significantly decreased in subchondral bone of UAC rats,and its co-location with osterix or dentin matrix protein-1 was identified in the subchondral bone region,where bone loss was significant.Conclusion: Egr1 reduction in PBLs potentially indicated subchondral bone OA lesions at early stage.