Radiographic Dynamic Observation Of Rabbit Knee Osteoarthritis And Changes Of Membrane Potential In Chondrocytes Isolated From Normal And Osteoarthritis Rabbit In Vitro Passage Culture

Knee osteoarthritis(OA)is a familiar chronic joint disease which is characterized by synovitis,degeneration of articular cartilage,remodeling of subchondral bone and arrophy of joint muscles.The clinicians have committed themselves to ameliorate the therapy of OA,but in general,the effect of the relavent treatment is not unexceptionable.The main reason is that OA can not be diagnosed in its early period and its pathogenesis is still unknown.In recent years,the techniques of imaging have become more and more important in diagnosis,analysis and treatment of OA.Radiography is the cheapest and widely used noninvasive examination.It could detect several OA features,including subchondral sclerosisi,osteophytes,cysts and joint space width.OA patients can be treated if OA is diagnosed early from X ray examination.Chondrocyte is the only one kind of cell type in articular cartilage and its biological change is closely related to the occurrence and development of OA.Although non-excitable cell,a large number of experiments confirmed that chondrocyte exits resting membrane potential(RMP).The functions of chondrocyte,such as cell volume,proliferation,differentiation and metabolism etc.,are sensitive to the changes of inside and outside ion concentration,and membrane potential affects the flow of these ions.It is important that clarifying membrane potential and its molecular mechanisms help us understand of the pathogenesis of OA.Lots of chondrocytes would be required in electrophysiological study of OA.The quantities of chondrocytes are limited and the cells are easy to differentiate in vitro,so it is generally believed that the culture of the first 3 to 5 passage cells can maintain the biological characteristics of chondrocytes.It is not clear how the electrophysiological properties,especially RMP will change during the passage of the normal and OA chondrocyte.In our experiments,Hulth method was established and the imagings of the knees in control and OA rabbits were examined by radiographic technique.The X-ray imaging dynamic analysis would help us explore the OA stage.Articular chondrocytes were enzymatic digested and subcultured in vitro to the 4th generation(named P1~ P4).The gene expressions of characteristic moleculars in chondrocytes were detected by qRT-PCR.RMP were recorded by patch clamp.Part 1 Radiographic dynamic observation of rabbit knee osteoarthritisObjectives: To explore the dynamic changes of articular images in osteoarthritis rabbits at different times.Methods: Hulth method in rabbits was established by using surgical techniques to create OA model.Non-invasive radiographs of the knee joints were performed in preoperative and postoperative 2 weeks to 12 weeks rabbits.We observe the configuration of the femorotibial joint and analyze the radiographic data by joint space ratios,joint gap angle and total number of osteophyte spur index.Results: The control group had normal performance of the knee joint during the 12 week times seen from the posterior-anterior radiographs.Compared to the preoperative knees,there were no significant changes of knee joints in postoperative 2 weeks and 4 weeks rabbits.Osteophytes began to appear from 6 weeks after operation,the joint spaces became narrow,especially in the lateral side and scleroses can be found in part of the surface of the joint.From 8 weeks to 12 weeks after operation,the narrows of joint space were getting more serious,the quantities of osteophytes were increasing and the scleroses of articular surface were more obvious.No abnormity could be seen by the lateral radiograph either in control or OA group.Compared to the preoperative knees,joint space ratios decreased,while joint gap angle and total number of osteophyte spur index increased in OA rabbtis from the 6th week after operation.Cnclusions: Hulth model rabbit is in the early stages of OA before postoperative 6 weeks,and gradually enter the middle and late stage of OA from 6 weeks after operation seem from radiographs.Part 2 Changes of membrane potential in chondrocytes isolated from normal and osteoarthritis rabbit in vitro passage cultureObjectives: To observe the changes of resting membrane potential(RMP)in chondrocytes isolated from normal and osteoarthritis(OA)rabbit in vitro passage culture.Methods: Hulth method in rabbits was established.Articular chondrocytes were enzymatic digested and subcultured in vitro to the 4th generation(named P1~ P4).The gene expressions of characteristic moleculars in chondrocytes were detected by qRT-PCR.RMP were recorded by patch clamp.Results: The P1~ P3 cells were ovoid or polygonal and P4 cells were fibroblast-like spindle shape in control and OA group.Compared to P1 cells of each group,the P2 ~ P4 cells showed a passage-dependent reduction of the mRNA expressions in collagen type II(COL2A1)and aggrecan(ACAN),and increase of that in collagen type I(COL1A1).Compared to control group P1 cells,the mRNA expression levels of COL2A1 and ACAN decreased,the value of RMP reduced significantly in OA group P1 cells.Compared to each group of P1 cells,the membrane potential values of P4 chondrocytes decreased in control group and increased significantly in OA group.Conclusions: The dedifferentiation phenomenon take place both in normal and OA chondrocytes,and RMP can serve as a description indicator of differentiation.Within 3 passages,their biological characteristics of chondrocytes can be kept,and they are suitable for cartilage tissue repair and the etiological study of OA disease.