The Study Of Regulatory Mechanism And Medical Imaging Of Internal And External Environment In Articular Cartilage

Background Since chondrocytes are non-renewable,damage of articular cartilage is difficult to repair.The early detection and treatment of damaged cartilage will improve prognosis,patient quality of life,and reduce economic burden.There are still many deficiencies and controversies concerning the detection and treatment of damaged cartilage.Synchrotron radiation and X-ray microscopy are help in microscopic imaging of soft tissue.The effects of mechanical environment and extracellular matrix structure on the metabolism of chondrocytes in articular cartilage are also the focus of orthopedic research.In order to effectively diagnose and treat cartilage disease,such as osteoarthritis,the understanding of cartilage functions and pathologies is required.The studies presented here not only explore the relationship between biomechanical properties and the structure of the extracellular matrix in immature,mature native and damaged cartilages by the new imaging technology,including X-ray phase-contrast imaging(PCI)and micro-computed tomography(micro-CT),but also investigate the gene target to inhibit apoptosis and cartilage degeneration in damaged cartilage.Objective The aims of this study were(1)to explore the imaging methods of synchrotron radiation X-ray PCI and micro-CT;(2)to determine the effects of collagen structure and contrast agent molecular mass on the diffusion property of contrast agent in fetal articular cartilage by X-ray PCI;(3)to measure the time-dependent and depth-dependent concentration change of contrast agent in native and damaged mature articular cartilage subjected to different dynamic loading parameters by micro-CT,and explore the relationship between the zone-dependent interstitial biomechanical function and the extracellular matrix structure in native and damaged cartilage;(4)to determine the potential role of microRNA-30b(miR-30b)in TNF-?-induced apoptosis,autophagy,and differentiation of the chondrogenic ATDC5 cell line.Methods(1)To determine the effects of molecular mass and collagen content on diffusion properties,two non-ionic contrast agents(iopamidol and iodixanol)were allowed to diffuse only through the surface of articular cartilage blocks(n=24)harvested from the femoral condyles of human fetuses(18~27 weeks gestation).The cartilage blocks were imaged using X-ray PCI before and after diffusion.The relationship between gray value and contrast agent concentration was calibrated so that gray value could be converted into concentration.The diffusion coefficients of iopamidol and iodixanol in human fetal articular cartilage were determined by fitting the experimental data to a one-dimensional finite element model.Differences of iodixanol and iopamidol diffusion coefficients in fetal articular cartilage blocks were analyzed by the two-tailed two independent sample t-test.A p-value of p<0.05 was considered statistically significant.(2)To understand the relationship between the zone-dependent interstitial biomechanical function and the extracellular matrix structure in native and damaged cartilage,full-thickness native(n=60 in 10 subgroups)and trypsin-degraded(n=60 in10 subgroups)mature articular cartilage blocks were studied by micro-CT.The cartilage blocks were fully immersed in Hexabrix and imaged by micro-CT before and after dynamic loading(5%and 15%strain;5,15,30,60 and 90 min loading time;0.5Hz loading rate).The depth-dependent glycosaminoglycan(GAG)concentration in native and trypsin-degraded cartilage was calculated based on Gibbs-Donnan equilibrium theory.The zone-dependent concentration change of Hexabrix was determined by calculating the area between the pre-loading and post-loading depth-concentration curves of Hexabrix for the superficial,transitional,and radial zones in native and trypsin-degraded cartilage using integral equations.For statistical analysis,two-tailed two independent sample t-test was used for comparing the GAG concentration between native and trypsin-degraded cartilage.One-way ANOVA with Tukey post-test was used for comparison among different groups,and between each group.A p-value of p<0.05 was considered statistically significant.(3)To determine the potential role of miR-30b in chondrocyte apoptosis,autophagy and differentiation,cell counting kit-8 and Hoechst 33342 staining were employed and the expression levels of caspase-3 and caspase-9 were assessed by western blot to analyze the effect of TNF-?on the viability of ATDC5 cells.Autophagy was examined by analyzing the levels of BECN1,ATG5,LC3B-II and p62 by western blot and quantitating GFP-LC3B by fluorescence microscopy.A luciferase reporter assay investigated the putative binding sites of miR-30b in the 3?-UTRs of BECN1 and ATG5 mRNAs.The effects of miR-30b and antimiR-30b on autophagy,apoptosis and osteogenic differentiation of TNF-?-treated cells were determined by autophagosome,apoptosis and alkaline phosphatase assays,respectively.Data were analyzed using the Student's t test between control group and treated group.One-way ANOVA with LSD test was used for comparison among different groups,and between each group.A p-value of p<0.05 was considered statistically significant.Results(1)In the diffusion property study,the turning time point before diffusion equilibrium of contrast agent in fetal articular cartilage is about one hour.The diffusion coefficient of iopamidol(3.25±0.71×10~-1010 m~2/s)was significantly greater than that of iodixanol(2.35±0.65×10~-1010 m~2/s)(p<0.05)in fetal articular cartilage,and the diffusion coefficients of both contrast agents did not have obvious tendency with gestational age.(2)In the biomechanics study,the GAG concentration was significantly decreased in trypsin-degraded cartilage compared to native cartilage(p<0.05),and the decrease of GAG was increasing with depth.For each loading time,the concentration changes of Hexabrix in each zone of the native and trypsin-degraded cartilage had no significant difference between 5%and 15%strain.The concentration changes of Hexabrix in superficial zone(SZ)and transitional zone(TZ)and radial zone(RZ)2 of native cartilage had no significant difference between each other in each loading time,but there was a significant difference between RZ1 and other zones which occurred within 5 min after loading(p<0.05).For trypsin-degraded cartilage,there was no significant difference between SZ and TZ in each loading time,but there was a significant difference between RZ2 and SZ/TZ which occurred after 30 min of loading(p<0.05),and there was still a significant difference between RZ1 and other zones which occurred within 5 min after loading(p<0.05).A significant difference was demonstrated for the concentration changes of Hexabrix in SZ and TZ between native and trypsin-degraded cartilage after 30 min of loading(p<0.05);there was a significant difference in both RZ1 and RZ2 within 5min after loading,and the changes were increasing with the loading time(p<0.05).(3)In the chondrocyte regulation study,TNF-?exposure increased apoptosis and positively regulated autophagy in ATDC5 cells.A direct interaction was detected between miR-30b and the mRNA 3?-UTRs of autophagy genes BECN1 and ATG5.The over-expression of miR-30b decreased TNF-?-induced autophagy and increased pro-apoptosis and extracellular matrix degradation in ATDC5 cells.However,treatment with antimiR-30b had the inverse effect.Conclusion These studies show that X-ray PCI and micro-CT provide a sensitive,highly resolved,and accurate method to respectively measure the diffusion property of contrast agent in immature cartilage,and time-dependent and depth-dependent concentration changes of contrast agent under dynamic loading in mature native and damaged cartilage.X-ray PCI has shown that the contrast agent molecular mass has an effect on their diffusion property in fetal articular cartilage,and the diffusion property of contrast agent is sensitive to the compositional and structural changes of extracellular matrix during cartilage maturation.Moreover,micro-CT has shown the flow field change of extracellular matrix not only concentrates in the upper radial zone of damaged cartilage,but also in the lower radial zone after dynamic loading.This change is sensitive to the compositional and structural changes of extracellular matrix.The upper radial zone of native mature cartilage may play an important role in pressure-bearing,and in damaged cartilage the lower radial zone may also make a contribution.Finally,we have found antimiR-30b can attenuate TNF-?-induced apoptosis and cartilage degradation by enhancing autophagy.These studies may provide a basis for further study the regulatory mechanism between biomechanical change and cartilage degradation,and for cartilage bionic,3D printing and other biogical materials.