| Objective: Temporomandibular joint disorder(TMD)is a common and frequently occurring disease in the oral and maxillary region.TMJ osteoarthritis(TMJOA)is the most common TMJ disease,characterized by progressive cartilage degeneration and subchondral bone sclerosis,which causes pain,stiffness,restricted mandibular movement,and affects patient quality of life.The etiology of TMJOA is complicated,and inflammation and abnormal subchondral bone remodeling are considered to be the most important pathogenic factors.Although extensive research has been carried out on the etiology of OA,there is no effective treatment for TMJOA.The best way to treat TMJOA is to improve the inflammation of the joint cavity,block the damage of condylar cartilage,and promote the degeneration of condylar cartilage and subchondral bone.Common treatments include surgery and non-surgical techniques.Non-surgical techniques are the most commonly used treatments for TMJOA,and include medications,physical therapies,and occlusal splints.Above treatments are effective in relieving symptoms,but long-term observation shows that the pathological effects on joint destruction are limited.Low-intensity pulsed ultrasound(LIPUS)is a non-invasive mechanical wave that can generate micro-mechanical stress in cells and tissues.It has been approved by FDA and widely applied in trauma,OA and other orthopedic diseases.The main studies of effects of LlPUS on OA are refered to articular cartilage,but there are few research on subchondral bone.Studies have demonstrated that significant subchondral bone changes occur following cartilage degradation in the condyle of TMJOA.Subchondral bone is considered a potential target in the treatment of OA.Transforming growth factor(TGF-β)pathway plays an important role in the pathological development of subchondral bone in OA and has been proved to be a potential target in the treatment of OA.A large number of studies have demonstrated that aberrant TGF-β signaling activity in subchondral bone contributes to the degeneration of articular cartilage with abnormal subchondral bone remodeling.It was found that inhibition of TGF-β activity prevents aberrant subchondral bone changes in OA.The aim of this study was to evaluate the effect of LIPUS on subchondral bone inTMJOA,and to investigate its effect on activation of the TGF-β1/Smad3 pathway and identify the therapeutic target of LIPUS.Studies were conducted in vivo and vitro and lay a foundation for clinical search for new treatment for TMJOA.Methods: 1.Mouse embryo osteoblast precursor cells(Mc3t3-E1)were induced by interleukin(IL)-1β.Enzyme linked immunosorbent assay method(ELISA)was used to detect the expression of IL-6 in cell supernatant level;The mRNA expression of matrix metalloproteinase(MMP)-13,TGF-β1 and Smad3 were detected by realtime fluorescence quantitative(qPCR).Phosphorylated Smad3(pSmad3)protein expression was detected by immunofluorescence staining.Western blotting was conducted to detect the protein expression of TGF-β1,type II receptor(TβRII),Smad3 and pSmad3.2.MC3T3-E1 cells were in the presence of IL-1β for 3 h.Antagonist of TGF-β1/Smad3pathway(SRI-011381)and transfecting with small interfering RNA(si RNA)of TβRII(si-TβRII)were used to activate and inhibit transduction of TGF-β1/Smad3 pathway in MC3T3-E1 cells respectively.Cells were grouped with or without LIPUS stimulation as follows: group A:(1)IL-1β;(2)LIPUS+IL-1β;(3)LIPUS+IL-1β+SRI-011381;group B:(1)si-NC+IL-1β;(2)si-TβRII+IL-1β;(3)LIPUS+IL-1β+si-NC;(4)LIPUS+IL-1β+si-TβRII.The expression level of IL-6 in cell supernatant was detected by ELISA.The mRNA expression of MMP-13,TGF-β1 and Smad3 in group A were detected by qPCR.Western blotting was conducted to detect the protein expression of TGF-β1,Smad3 and pSmad3 in both of the two groups.3.Rabbit TMJOA model was established by type II collagenase induction.The left joint in this model was continuously stimulated with LIPUS for 3 and 6 weeks(1 MHz,30 mW/cm2)for 20 min/day.The morphological and histological features of subchondral bone were respectively examined by Micro-computed tomography(Micro-CT)and Safranin-O staining.The number of osteoclasts was quantitativly assessed by tartrate-resistant acid phosphatase(TRAP)staining.Immunohistochemistry and western blot analysis were conducted to evaluate the protein expression of Cathepsin K and TGF-β1/Smad3 pathway.Results: 1.IL-1β induced cells for 3 h can simultaneously cause significantly increases in the production of IL-6 in cell supernatant,the mRNA expression levels of MMP-13 and activity of TGF-β1/Smad3 pathway.2.Agonist(SRI-011381)and si-TβRII transfection significantly activated and inhibited the transduction of TGF-β1/Smad3 pathway respectively.TβRII knockdown significantly decreases the expression of IL-6 induced by IL-1β comparing.This result suggested that inhibition of TGF-β1/Smad3 pathway attenuated inflammatory cytokines expression in IL-1β induced MC3T3-E1 cells.LIPUS stimulation significantly reduced IL-6,the relative mRNA expression level of MMP-13 and the activation of TGF-β1/Smad3 pathway.The inhibitory effect of LIPUS on the pathway was counteracted by the induction of agonist and si-TβRII transfection.3.LIPUS could improve the trabecular microstructure and histological characteristics of subchondral bone in rabbit TMJOA.It also suppressed abnormal subchondral bone resorption and activation of TGF-β1/Smad3 pathway,characterized by the number of osteoclasts,protein expression levels of Cathepsin K,TGF-β1,TβRII and pSmad3 were decreased.Conclusions: 1.IL-1β caused inflammation and activated TGF-β1/Smad3 pathway in MC3T3-E1 cells.2.Inflammation was inhibited by suppressing TGF-β1/Smad3 pathway.3.LIPUS ameliorated inflammation by inhibition of TGF-β1/Smad3 pathway.4.LIPUS improve the quality of subchondral bone in rabbit TMJOA by suppressing abnormal osteoclast activity and TGF-β1/Smad3 pathway.5.TGF-β1/Smad3 pathway may be a potential target of LIPUS therapy in the treatment of TMJOA.6.LIPUS is a potential therapeutic treatment for TMJOA. |
PDF Full Text Request