Exploratory Study On The Cellular Metabolic Pathway Of Goat Temporomandibularjoint Disc

Background:The Temporomandibular Joint?TMJ?is one of the most complex joints in the human body.It plays an important role in the process of chewing,swallowing,and pronunciation in the oral and maxillofacial.Temporomandibular disorders?TMD?are a group of degenerative diseases of the musculoskeletal system associated with morphology and function.The degenerative changes of the temporomandibular joint are closely related to the structural integrity of the articular disc.Among them,the joint disease characterized by irreversible lesions such as thinning of the articular disc,transparent deformation,tear and perforation accounts for about 5%to 10%of the total TMD.In recent years,tissue engineering research has provide a new way to replace irreversible damaged TMJ disc tissue.When the goat temporomandibular joint disc self-assembly tissue engineering technology is constructed,it is found that the volume of tissue globules after one week of inoculation is obviously contracted during the self-assembly matrix culture,and the contraction of the self-assembled matrix is not conducive to the production of extracellular matrix of the articular disc and the growth of the disc substrate.At the same time,studies on its mechanism have also shown that cellular metabolism may be involved in the pathophysiological processes.Faced with this problem,the research team believes that it is necessary to study the physiological metabolic process of TMJ disc cells.The research on the energy metabolism pathway of TMJ disc cells may provide help and more basis for clarifying the above problems.Objective:The purpose of this study was to explore the metabolic pathways of the different hypoxia and glucose culture on goat temporomandibular joint cells so as to provide a clearer realization of the physiological and biochemical environment and metabolic mechanism of goat temporomandibular joint cells.Furthermore,to lay foundation for the prevention and treatment of TMJ disease as well as tissue engineering.Methods:The goat temporomandibular joint disc cells in vitro were isolated and passaged to the P2 generation.The P2 cells were cultured in 21%O₂&complete medium named by group A while 2%O₂&complete medium named by group B.The cells were cultured in 21%O₂&sugar-free medium named by Group C as well as 2%O₂&sugar-free medium named by Group D.In addition,the cells cultured in 21%O₂+complete medium?Group A?was used as a control.At 12h,24h,36h,48h,and 60h after culturing in their culture environments,the indicators were tested respectively.Morphological changes of goat articular disc cells on different cultures were observed by fluorescent inversion microscope.The growth and proliferation of goat articular disc cells were detected by CCK8 assay.The apoptosis of goat articular disc cells was detected by hoechst 33258 staining assay.Cellular lactate dehydrogenase?LDH?,cellular reactive oxygen species?ROS?and superoxide dismutase?SOD?were detected by enzyme-linked immunosorbent assay?ELISA?.The Mitotracker staining experiment was used to observe the mitochondria in goat articular disc cells under different environments.Results:1.The fluorescent inversion microscope showed that most cells in control group?treated with 21%O₂&complete medium?became spindle-shaped at each time point?12h,24h,36h,48h,60h?.After glucose deprivation,the cell morphology was in contrast to the control group.In comparison,a large number of cells were changed from long fusiform to polygonal,and under a single hypoxic condition,only a small number of cells changed from long fusiform to polygonal.2.Proliferation and apoptosis test indicated that the cells cultured in the 2%O₂&complete culture medium had better proliferation compared with the control group,and after glucose deprivation,the proliferation of the cells was significantly decreased under both normoxia and hypoxia conditions?p<0.05?.After 24 hours of sugar-free culture,the cell proliferation rate was negative.The results of Hoechst 33258 staining showed that compared with the control group,the cells in each sugar deprivation group showed obvious apoptosis,especially after 24 hours of sugar-free culture,the apoptosis rate continued to increase.The apoptosis rate of the cells in the 2%O₂&complete medium group was lower than that in the control group?p<0.05?.This is corresponding to the CCK8 experimental results.3.Intracellular LDH results showed that the the control group?21%O₂?began to decrease from 12h.The hypoxia group,the glucose deprivation group,and the hypoxia&glucose deprivation group all showed a trend of increasing first and then decreasing,and peaking at 24h.The overall comparison of the four groups showed that only the concentration of LDH in the hypoxia group was higher than the control group,and the concentration of the two groups after glucose deprivation was lower than that of the control group,and the concentration of LDH in the glucose deprived group was the lowest?p<0.05?.4.Intracellular ROS results showed that the ROS in the control group increased first but decreased afterwards,and reached peak at 36h.The 2%O₂ group showed a tendency to rise first,then fall,and rose again.The glucose deprivation group first decreased and then rose,and 36h was the minimum.The 2%O₂&glucose deprivation group was consistent with the control group?p<0.05?.The overall comparison of the four groups showed that the concentration of ROS in the control group was higher than the other three groups,and the ROS concentration in the glucose deprivation group was the lowest?p<0.05?.5.The intracellular SOD results showed that the SOD in the control group?21%O₂?decreased first and then increased,and the 48h was minimum value.The2%O₂ group showed a trend of decreasing first,then rising,and falling again.The sugar deprivation group showed a continuous downward trend.The 2%O₂&glucose deprivation group showed a continuous upward trend?p<0.05?.The overall comparison of the four groups showed that only in the 2%O₂ group,the intracellular SOD concentration was higher than that in the control group,while in the two groups after glucose deprivation,the intracellular SOD was lower than the control group?excluding the 2%O₂&glucose deprivation group at 12h?.Among them,the intracellular ROS concentration in the glucose deprivation group was the lowest?p<0.05?.In addition,the concentration of SOD in each group is always negatively correlated with the presence of ROS.6.Mitotracker staining results showed that the number of mitochondria in the control group?21%O₂?began to increase from 12h,and began to decrease after 48h.Compared with the control group,the number of mitochondria in the three groups was small,especially in the two groups after glucose deprivation,the number of mitochondria decreased significantly,and the glucose deprivation group showed a first increase and then a downward trend,24h was the high value;2%O₂&glucose deprivation group,the number of mitochondria first rise and then fall,36h was the peak.However,no significant changes in mitochondrial number were observed in the hypoxic group.Overall,the number of mitochondria in the glucose deprived group was the lowest?p<0.05?.This was basically consistent with the ROS test results.Conclusion:2%O₂ was more conducive to the metabolism of goat temporomandibular joint disc cells.Glucose deprivation had a significant down-regulation of goat temporomandibular joint disc cells;Goat TMJ disc cells had an early adaptive response to 2%O₂&glucose deprivation.However,their metabolic capacity declined in the end.