The Establishment Of The Traumatic TMJ Ankylosis Animal Model And Screen Related Molecules

Temporomandibular joint(TMJ)ankylosis is a serious oral maxillofacial disease which causes healthy problems physically and mentally.It often causes difficulties in opening mouth and chewing.Among these numerous pathogenic factors,trauma is the most common reason for TMJ ankylosis.However,the mechanism under TMJ ankylosis is still unclear.Our previous study has already confirmed that the lateral pterygoid muscle(LPM)plays a role in the occurrence of traumatic TMJ ankylosis as distraction osteogenesis.But its molecular mechanism is still unclear.This study has established a traumatic TMJ ankylosis model by compositing trauma which based on the susceptibility factors of traumatic TMJ ankylosis.We compared the gene expression between normal and LPM broken TMJ ankylosis models by gene chip and RT-q PCR to verify that the molecule differences are consistent with distraction osteogenesis and the role of the lateral pterygoid muscle in traumatic TMJ ankylosis.Part one: The establishment of the traumatic TMJ ankylosis animal modelObjective: To establish a traumatic TMJ ankylosis model through the composite trauma and to provide the basis for later study.Methods: One sheep was set as normal control.The other 8 sheep were randomly divided into 12-week and 24-week groups to perform the joint operation respectively.The operation consisted of condylar sagittal fracture,outside 1/4 articular disc excision and cross deep grooves.After 12 weeks or 24 weeks,the animals were sacrificed.Gross observation and spiral CT were used to evaluate the condylar morphology.Results: In the experimental group,fibrous ankylosis occurred at the 12 th week and bony ankylosis occurred at the 24 th week.The globular complex in condylar could be observed after 12 weeks.Meanwhile,the condylar diameter(anteroposterior diameter,1.20±0.10 cm vs 0.45±0.03 cm,inside diameter,2.64±0.12 cm vs 1.75±0.03 cm)was increased(P<0.05)and the maximum mouth open degree(2.48±0.33 cm vs 4.65±0.38 cm)was reduced(P<0.05).The CT scanning outcomes suggested that both bone formation and bone resorption existed.Condylar globular complexes were more obvious at the 24 th week and the osseous adhesion was visible.Condylar diameter(anteroposterior diameter,1.65±0.12 cm vs 1.20±0.10 cm,inside diameter,3.55±0.12 cm vs 2.64±0.12 cm)was further increased(P<0.05)and mouth open degree(1.80±0.16 cm vs 2.48±0.33 cm)was further reduced(P<0.05).More bone tissue can be seen in the joint space.Conclusions: The compound trauma can successfully establish traumatic TMJ ankylosis model and it causing the osseous stiffness of the condylar area.Part two: The traumatic TMJ ankylosis related molecules verification Experiment one: Gene chip screening of the traumatic TMJ ankylosis related moleculesObjective: Through the high-throughput microarray technology to verify traumatic TMJ ankylosis related molecules.Methods: The sheep were randomly divided into4-week and 12-week groups.Each group had 1 normal sheep without any surgery.Except the normal controlled sheep,all the others had operation.The left side of the sheep’s condylar was subjected to the operation according to the first part of this experiment,while the right condylar undergone both the condylar modeling operation and LPM broken.After 4 and 12 weeks,the sheep were executed and the bone of condylar fracture zone was sent for gene chip analysis.The clustering heat maps,scatter plot,DAVID enrichment analysis,functional hierarchical network platform,GO build(GO – network)and Pathway analysis were used for further analysis in order to find out the related molecules.Results: After 4 weeks,the index of bone morphogenic factors,muscle related factors and osteoclast related factors increased in the non LPM broken groupas well as the LPM group.However,the increase of the LPM broken group was lower than the non LPM broken group.After 12 weeks,the related factors in non LPM broken group were also higher than the LPM broken group.This phenomenon existed even after 12 weeks.Compared with the 4-week group,the index of muscle related factors was decreased in the12-week group while the index of ossification factors were increased in non LPM broken group.The bone morphogeneic factors and ossification factors were increased in the LPM broken group.Through a variety of analyses,the following factors were selected to be related factors,namely,TGF-β1,COL2A1,OCN,LECT1,BMP7,FOS,CALCR,IL1 A,COMP,CLEC3 A.Conclusions: The distraction effect from LPM can promote osteogenic related molecules expression in ankylosis area,which is similar to distraction osteogenesis.The broken LPM can effectively restrict the expression of osteogenic molecules,which was similar to normal bone fracture healing process.TGF-β1,COL2A1,OCN,LECT1,BMP7,FOS,CALCR,IL1 A,COMP,CLEC3 A may play important roles in the osteogenesis of traumatic TMJ ankylosis.Experiment two: Confirmation of TMJ ankylosis related molecules by RT-q PCRObjective: To confirm the validated traumatic TMJ ankylosis related molecules of the experiment one.Methods: the animal preparation was the same as experiment one.Each group had 1 normal sheep without surgery.After 4 and 12 weeks,the animals were executed,the BMP7,CALCR,FOS and LECT1 expression were analyzed by RT-q PCR.Results: The expressions of BMP7 and LECT1 corresponded to gene chip data.CALCR expression was the same with gene chip only in 4-week group.While the FOS expression was consistent with the operation process competently.Conclusions: The expression of osteogenic molecules of non LPM broken group consisted with the distraction osteogenesis group,while the expression of these osteogenic molecules in LPM broken group were much similar to normal bone fracture.These results showed that the function of lateral pterygoid muscle making the distraction osteogenesis,leading to the TMJ ankylosis by excessive bone formation in the fracture area.