| Background:Osteoarthritis(OA) of knee is the most common clinical chronic degenerative joint disease, seriously affecting the quality of human life. The risk factors and progress mechanism of OA is not clear. In recent years, many studies have shown that OA is considered a disease of the entire joint, involving all joint tissues. Bone and cartilage composite unit, constitute by articular cartilage and subchondral bone, and its plays a very important role in structural and functional change on the development of OA. The bone and cartilage composite unit of OA exists biochemistry and molecular interactions. This study was to investigate the cartilage and subchondral bone pathology, angiogenesis, mechanical properties, molecular genetic level, permeability of the human primary knee OA and post-traumatic OA, and to evaluate the change and interaction of bone and cartilage composite unit between different stage of OA.Methods:1.The human tibial plateaus were obtained from patients undergoing total knee arthroplasty (TKA),from October2012to Match2013. After gross observation, the cartilage-bone complex samples were taken out from the most weight-bearing regions in the internal areas of the medial and lateral plateaus by and divided into A, B, C, D four parts. A group was evaluated the severity of subchondral bone by Micro-CT scanning system, and stained with histological staining (HE staining, safranin O/fast green staining, toluidine blue staining) and Makin scores, immunohistochemical staining. B group was conducted cartilage point pressure, nano-indentation and other biomechanical testing. C group was processed real-time quantitative PCR and Westernblot detection. D group was proceeded contrast agents to strengthen scanning method under CT to detect the permeability.2.Thirty New Zealand white rabbits were randomly divided into two groups:Anterior cruciate ligament transaction (ACLT) group (n=15); control group (n=15). OA was surgically induced in right knee. The rabbit knees were evaluated by gross observation, histological examination, immunohistochemistry, and vascular density number detection was done at4,8and12weeks after ACLT. Histological valuation was performed with safranin-O staining and OARSI score, immunohistochemistry, vascular density was performed to confirm the vascular invasion at osteochondral junction, real-time quantitative PCR.Results:1. According to the Makin scores and pathology detection, Stage I was characterised by fissures within the superficial zone and duplicated tidemark; Stage II was charactrised by cartilage erosion,mircocracks and vascular invasion into calcified zone; Stage III was characterised by multiple fissures extending into the subchondral bone, subchondral cysts, vessels across tidemark and obviously thickend subchondral bone; Stage IV was characterised by full-thickness cartilage defects, endochondral ossification, and naked subchondral bone. The bone histomorphometric study showed that bone mineral density(BMD), bone volume(BV)/tissue volume(TV), tissue mineral density(TMD), trabecular number(Tb.N) and trabecular thickness (Tb.Th) in later stage were significantly higher than that of the early stage while Tb.Sp and SMI were lower (P<0.05), there were statistically significant differences between each stage(P<0.05). The severity of articular cartilage degeneration, as assessed by Makin scores,was significantly correlated with BMD (r=0.83,P<0.001), BV/T V (r=0.79,P<0.001) and Tb.Th(r=0.65,P<0.001), and inversely correlated with SMI(r=-0.76, P<0.001). Mechanical test results showed that stage III was the lowest elastic modulus of OA cartilage, and stage IV was the maximum bone elastic modulus, hardness of the OA cartilage. Immunohistochemistral results showed that RANKL was expressed positive in stage II, VEGF was expressed positive in stage II and III, Runx2and OPG are expressed positive in stage III. The PCR showed that osteogenesis-related genes Runx2, OCN, OPN are the highest expressed in stage III. Osteoclast-related genes Cathepsin K, RANKL are the highest expressed in stage â…¡, VEGF was the highest expressed in stage â…¢. Chondrocyte differentiation related genes Aggrecan, Sox9, COLII are downward trend, and are the highest expressed in stage â… . Cartilage damage associated gene MMP9was upward trend, and was the highest expressed in stage â…£. Westernblot showed that osteoblast-related proteins Runx2, BMP2, BMP7, OPG are the highest expressed in stage â…¢. Osteoclast-related proteins RANK, RANKL are the highest expressed in stage â…¡. Permeability results showed that the permeability of cartilage was associated with cartilage degeneration, and the penetration rate of stage â…¢ was the fastest.2. Rabbits in ACLT group after4weeks showed a typical OA pathological changes, including loss of surface integrity, fissuring and microcracks, loss of cartilage SO stain, naked subchondral bone increased at8and12weeks. OARSI score in different time point were statistically significant (P<0.05) compared with the control group. VEGF immunohistochemical staining indicated the ACLT group positive results, and the highest expressed at12weeks. Vascular invasion numbers significantly increased in ACLT group compared to control group and the highest density of blood vessels at12weeks, and there were statistically significant differences between each time point (P<0.05). PCR showed that VEGF was the highest expressed at12weeks.Conclusions:1. The interactions of bone and cartilage composite unit is crucial in OA progression. The subchondral bone remodeling is closely related with cartilage degeneration. The osteoclasts are active in early stage, and osteoblasts are active in late stage of OA. The contrast-enhanced scanning method based on CT can detect the permeability of cartilage and reflecting the cartilage degeneration.2. Angiogenic activity of subchondral bone was elevated in the early of progressive stage of OA and associated with interaction. The degree of vascular invasion into the osteochondral junction increased time-dependently with the progressive stage of OA. |
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