| Traumatic temporomandibular joint(TMJ)ankylosis is a disease that seriously affects the function and physiology morphology of the oral and maxillofacial system,characterized by restricted mouth opening and difficulty in chewing.There are many hypotheses about the pathogenesis of traumatic TMJ stiffness,but none of them can fully explain the mechanism of its occurrence.Previous studies on animal models of traumatic TMJ ankylosis have shown that sheep,due to their strong immunity,similar TMJ size,and anatomical structure to humans,are the most commonly used experimental animals for studying traumatic TMJ ankylosis.However,sheep are unsuitable for studying the pathogenesis of traumatic TMJ ankylosis because of the difficulty of gene manipulation and the few antibodies that can be applied to sheep.Although transgenic mice are easy for gene manipulation,the size of the mouse TMJ is too small to simulate TMJ trauma surgery.In contrast,rats have the advantages of both surgical simulating trauma surgery and being genetically manipulable.In the pathogenesis of traumatic TMJ ankylosis,macrophages regulate cartilage callus formation in the early stages of healing after TMJ injury.In the pathogenesis of traumatic TMJ ankylosis,macrophages are closely related to TMJ ankylosis.Our previous research found that chondrocyte ossification is obvious in sheep TMJ bony ankylosis in the early stage,and the only cell type in cartilage tissue is chondrocytes,and cartilage ossification must undergo hypertrophy of chondrocytes.Therefore,chondrocyte hypertrophy and traumatic TMJ bony ankylosis are closely related.Based on the aforementioned research facts and analysis,this project intends to use compound trauma to model SD rat traumatic TMJ ankylosis animal model,and use methods such as gross observation,Micro-CT imaging,and histological examination to verify the animal model and determine the type of TMJ ankylosis.The aim is to construct a stable,highly reproducible,and easily scalable SD rat model of traumatic TMJ ankylosis.Using the SD rat traumatic TMJ ankylosis animal model,RNA-seq will be used to detect the total RNA of the SD rat TMJ complex in the postoperative control group and each experimental group,and to find the key factors and signaling pathways that affect the traumatic TMJ ankylosis.The bone marrow-derived macrophages and primary condylar chondrocytes of SD rats were isolated and cultured,and the conditioned medium of M1 macrophages and M2 macrophages were added to the primary condylar chondrocytes.Western Blot,RT-qPCR,and other molecular biological methods will be used to reveal the molecular mechanism by which macrophages affect condylar chondrocyte hypertrophy,providing experimental evidence for clarifying the pathogenesis of traumatic TMJ bony ankylosis.Part 1: Establishment of traumatic temporomandibular joint ankylosis model in SD rats Objective: To establish an SD rat modal of traumatic TMJ tonicity and verify the type of tonicity,laying the foundation for animal studies to clarify the pathogenesis of this disease.Methods: Sixty 3-week-old male SD rats were selected and randomly divided into experimental and control group.The left TMJ of the experimental group rats was defined as the "operated side" and subjected to simulated TMJ trauma,while the right TMJ was defined as the "sham operation side" and subjected to sham surgery.The SD rats in the experimental group underwent skin preparation and disinfection on the temporal and preauricular regions under general anesthesia.On the sham-operated side of SD rats in the experimental group,a preauricular incision was made,bluntly dissected to the joint space,and then the wound was sutured in layers.The SD rats in the experimental group underwent surgery to induce TMJ ankylosis.A 1 cm long preauricular incision was made to separate and expose the joint capsule and articular disc.Part of the articular disc was excised and part of the condylar fibrocartilage and glenoid fossa were damaged,and suturing the wound in layers.The control group rat did not undergo any surgery.The vertical passive maximum mouth opening and mandibular lateral deviation in the experimental group and control group of rats were measured at 1 week,4 weeks,and 8weeks after operation.Gross observation,Micro-CT,and histology were used to judge whether TMJ stiffness occurred in SD rats,and to determine the type of stiffness.Results: Compared with the control group,the vertical passive maximum mouth opening of the SD rats in the experimental group continued to decrease at 1 week,4 weeks,and 8weeks after the operation,and the lateral opening of the mandible continued to increase.Gross observation showed that the TMJ on the surgical side in the experimental group fused and enlarged 8 weeks after the operation,and the joint space disappeared.Compared with the sham-operated side,the micro-CT results showed that bony fusion occurred at the TMJ on the operated side,the joint space disappeared,and a large number of calcified callus formed.Histological examination showed that endochondral ossification existed in the TMJ on the surgical side of the experimental group.Conclusion: The animal model of traumatic TMJ ankylosis in SD rats was successfully established,and the type of ankylosis was determined to be bony ankylosis,and it was preliminarily confirmed that there was chondral ossification in the pathogenesis of traumatic TMJ ankylosis in SD rats.Part Ⅱ: Macrophage-mediated hypertrophy of condylar chondrocytes involved in temporomandibular joint bony ankylosis in SD rats Objective: To clarify the role of macrophage and chondrocyte hypertrophy in the progression of traumatic TMJ ankylosis in SD rats.Method: A control group was set up,and total RNA of TMJ complex of SD rats from the successfully modeled SD rats at 1 week,4 weeks,and 8 weeks after the operation,detect the RNA concentration,and submit it to Huada Gene Company after completing the RNA-seq detection.BGI Group compared the sequencing data with the rat reference gene bank(NCBI,GCF＿000001895.5＿Rnor＿6.0)based on the BGISEQ-500 platform.Analyze the raw data and find out the key factors and the most significantly changed signaling pathways,and verify the RNA-seq detection results by RT-qPCR and immunofluorescence.Results: A total of 38,554 changed genes were detected by RNA-seq.There were 3,453 differentially expressed genes between the experimental group and the control group at 1week after the operation,and 450 differentially expressed genes between the experimental group and the control group 4 weeks after the operation.There were 2939 differentially expressed genes between the group and the control group at 8 weeks after the operation,and the differentially expressed genes in the above three groups were found,and 25 differential genes were obtained.According to GO analysis and KEGG analysis,it was found that IL-1β was the key factor,and the change of PI3K/AKT signaling pathway was the most significant.The chondrocyte hypertrophy-related factors ALP,Col X,MMP13,and RUNX2 were highly expressed in the 4-week postoperative group,and the M1 macrophage marker CD86 was highly expressed in the 4-week postoperative group by immunofluorescence.Conclusion: Macrophages are involved in the development of traumatic TMJ ankylosis in SD rats,and the PI3K/AKT signaling pathway appears to play a key role.Part Ⅲ: M1 macrophages regulates condylar chondrocyte hypertrophy via PI3K/AKT pathway by paracrine secretion of IL-1β.Objective: To clarify the mechanism by which M1 macrophages regulate chondrocyte hypertrophy via PI3K/AKT pathway by paracrine secretion of IL-1β.;Methods: Three-week-old male SD rats were selected,primary condyle chondrocytes were isolated and cultured in vitro,and identified by type Ⅱ collagen immunohistochemistry and Alcian blue staining.The bone marrow-derived macrophages of SD rats were isolated and cultured in vitro,the primary M0 macrophages were obtained and identified by flow cytometry,and the M0 macrophages were induced to polarize into M1 macrophages and M2 macrophages respectively.The phenotypes of M1 macrophages and M2 macrophages were identified by RT-qPCR and WB analysis.The conditioned medium of M1 macrophages and M2 macrophages were obtained,and add condyle chondrocytes to the culture,simulate the microenvironment of TMJ tonic,use RT-qPCR and WB to detect the changes of PI3K/AKT signaling pathway,and inhibit PI3K/AKT after pathway,the hypertrophy-related factors of chondrocytes changed.Results: Primary rat condylar chondrocytes were positive for Coll Ⅱ and Alcian blue staining.The pro-inflammatory markers IL-1β and i NOS genes and proteins in M1 macrophages were highly expressed,and the anti-inflammatory markers Arg-1 and CD206 genes and proteins in M2 macrophages were highly expressed.After M1 macrophage conditioned medium was co-cultured with condylar chondrocytes,the PI3K/AKT signaling pathway was activated,and chondrocyte hypertrophy-related genes such as ALP,Col X,MMP13,and RUNX2 were significantly up-regulated.But M2 macrophage conditioned culture Genes can not activate PI3K/AKT signaling pathway,nor can promote the expression of genes related to chondrocyte hypertrophy.When GIBH-130(IL-1βinhibitor)was added to the conditioned medium of M1 macrophages,the PI3K/AKT signaling pathway and chondrocyte hypertrophy-related genes were inhibited.Subsequent inhibition of either PI3 K or AKT resulted in down-regulation of chondrocyte hypertrophyrelated genes.Conclusion: M1 macrophages activate PI3K/AKT signaling pathway and regulate chondrocyte hypertrophy via paracrine secretion of IL-1β,thereby positively regulating the bone ankylosis of traumatic TMJ.In summary,this study used TMJ compound trauma to construct an animal model of SD rat traumatic TMJ ankylosis,verified the reliability and stability of the model,and determined that the ankylosis type of SD rat traumatic TMJ ankylosis animal model is bony ankylosis,there is chondral ossification in the process of TMJ bony ankylosis.Total RNA was extracted from the TMJ complex at multiple time points after surgery and subjected to RNA-seq sequencing performed.It was preliminarily determined that IL-1βwas the key factor,and PI3K/AKT was the signaling pathway with the most significant changes.Through histological and in vitro experiments,it was determined that M1 macrophages induce condylar chondrocyte hypertrophy via paracrine secretion of IL-1βthrough PI3K/AKT signaling pathway,thereby aggravating traumatic TMJ bone ankylosis in SD rats. |
PDF Full Text Request