The Mechanism Of The Interaction Between Articular Cartilage And Subchondral Bone In Osteoarthritis

Objective Osteoarthritis(OA)is often accompanied by degeneration of cartilage and pathological changes of the subchondral bone,during which development,chondrocytes and osteoblasts play an important role.This study aimed to investigate the mechanism of chondrocyte-osteoblast interaction in vitro and explore the mechanism of occurrence and development of pathological changes of articular cartilage and subchondral bone with different exercise intensity in osteoporosis and arthritis models.Methods1.In vitro,osteoblasts and chondrocytes were obtained by enzymatic digestion from within 24 h of birth and 4w of SD rats,respectively.Osteoblasts were cultured using conditioned medium of chondrocytes for alkaline phosphatase staining.Rat chondrocytes induced by IL-1? were indirectly co-cultured with osteoblast conditioned medium.Western blotting was used to detect the expression of cyclooxygenase COX-2,MMP-13 and ADAMTS-5.Meanwhile,Western blotting method was used to detect the activation of OPG/RANK/RANKL,MAPK,NF-?B and other pathways.2.In vivo,60 female SD rats of 12 weeks were selected for ovariectomy.Then anterior cruciate ligament resection of the knee was performed after the operation.After surgery,rats were divided into five groups: the shamFree group was sham-operated for anterior cruciate ligament resection,the group without intervention was Free group,the low-speed running exercise(12m /min)was L-TW group,the high-speed running exercise(18m/min)for the H-TW group,and subcutaneous injection of bisphosphonates [30?g /(kg· d)] after surgery was used as anti-OP group.At the 4th and eighth weeks after the grouping,the CTX-?,CTX-?and OPG proteins in the blood and urine samples of ratswere detected by ELISA.The knee joints were scanned by micro-CT instruments.And the rat femoral paraffin sections were stained with hematoxylin-eosin,toluidine blue and Safranin-O/fast green and were subjected to immunohistochemical staining such as type?/?/ ? collagen.Osteoarthritis cartilage pathology OARSI scoring system was used for scoring of the femurs.Results1.Rat chondrocytes and osteoblasts were successfully obtained in vitro and their phenotypic characteristics were maintained.In the indirect co-culture system of chondrocytes and osteoblasts in vitro,chondrocyte conditioned medium may promote the synthesis of osteoblast alkaline phosphatase,while osteoblast conditioned medium may up-regulate the inflammatory index of inflammatory chondrocytes COX-2 and proteoglycanase ADAMTS-5 expression,down-regulate the expression of matrix metalloproteinase MMP13.Meanwhile,osteoblast conditioned medium can activate and up-regulate NF-?B / P65,MAPK-P38 / JNK / ERK and other signaling pathways.2.The ovariectomized rats could cause osteoporosis and instability of the knee joint after anterior cruciate ligament resection.At the 4th week,CTX-? increased significantly in the blood of H-TW group and L-TW group,meanwhile OPG in H-TW group increased significantly.At the eighth week,the level of CTX-? in H-TW group was significantly decreased,while the level of OPG in blood was significantly increased in H-TW group and anti-OP group,but significantly decreased in urine,and the concentration of CTX-? in urine was significantly increased.At the 4th week,the amount of bone in the lower femur of H-TW group and anti-OP group increased significantly,and the effect of bisphosphonate decreased at the eighth week.At the eighth week,the osteogenic effect of subchondral bone in both groups was obvious.Compared with L-TW group and H-TW group,anti-OP group showed no significant changes in articular cartilage thickness,cell morphology and articular surface,and the toluidine blue stain deepened.However,in the L-TW group andthe H-TW group,the articular cartilage layer was significantly reduced,the morphology of the surface chondrocytes was changed,the concavity and convexity disappeared,the activity of the subchondral bone was enhanced,and the tideline was changed.In the H-TW group,articular surface roughness,chondrocyte reduction accompanied by fibrosis,Toluidine blue uneven coloring.For the glycosaminoglycan and type II / X collagens staining,the L-TW group stained more clearly,while the high-intensity exercise H-TW group lighter.OARSI score,H-TW group scores were significantly higher than Free group,p <0.05.Conclusion1.In the indirect co-culture system of chondrocytes and osteoblasts in vitro,the phenotypes of chondrocytes and osteoblasts are changed.The chondrocyte conditioned medium may promote the osteoblast bone formation,while osteoblast medium can change the inflammatory index of inflammatory chondrocytes or the expression of matrix enzyme.In addition,MAPK,NF-?B / P65 signaling pathway may play a crucial role in the above process.2.After anterior cruciate ligament resection in menopausal rats,early high-intensity high-speed running exercise and anti-osteoporosis with bisphosphonates can enhance the bone mass of the lower femur,reverse osteoporosis after ovariectomy,but also can significantly increase the amount of bone in the subchondral bone of the knee to enhance osteogenesis.At the eighth week after anterior cruciate ligament resection in rats of menopause,high-intensity running exercise can significantly accelerate the course of arthritis.Low-intensity running exercise significantly inhibited or delayed the degeneration of articular cartilage.Supplementary bisphosphonates do not accelerate the degeneration of articular cartilage.