The Permeability Of Calcified Cartilage Zone And The Mechanism Of Its Development Induced By T-VDCC

BackgroundOsteoarthritis(OA)is a common degenerative disease in clinic,which often leads to joint pain,limited activity and even joint disability.OA is a complex disease,but it is a mutual understanding that OA is inflammatory disease affecting the whole synovial joint.However,it is not clear how the inflammation exaggerated and where it began.Increasing studies have shown that the cause of OA may come from the subchondral bone,which originated from the slight inflammation.Without intervention,it continued to cause the inflammation in cartilage,thus the positive feedback cycle of inflammation formed,which may eventually lead to the occurrence and development of OA.If this cycle could be interrupted in early time,it may release the process of OA greatly.In physiological condition,cartilage and subchondral were in two different micro environments and there is no communication between them.It is commonly accepted that cartilage is avascular,aneural,and hypoxia,while bone is rich in blood supply and aerobic.Calcified cartilage zone(CCZ)is a dense boundary layer between cartilage and subchondral bone,and the upper part is connected with cartilage by tide mark,and the lower part is connected with the subchondral bone by cement line.The special morphology and composition of CCZ give the tissue unique mechanical properties,which attenuated load forces on cartilage during the movement,and also can transform the shear force to compressive stress from cartilage to subchondral bone.In addition,CCZ also was an important barrier,which would rise abnormal cross-talk between bone and cartilage when absent,resulting in triggering OA.However,we know little about this field and the previous research results are controversial.Therefore,we should use various technologies and models to explore the barrier function of CCZ.But if we try to understand the various functions of CCZ,we must start with the formation and development of CCZ,so as to understand the mechanism of remodeling in pathology.In the development of CCZ,we speculated that it also experienced a similar process of endochondral ossification,but this process is different from the endochondral osteogenesis in growth plate.However,in the process of cartilage transformed to bone(calcified cartilage),chondrocyte hypertrophy,apoptosis and differentiation are the key points in the process.A lot of evidence shows that voltage dependent calcium channel(VDCC)is widely expressed in various osteochondrogenic phases,and their spatiotemporal distribution plays a decisive role in the osteochondral development.Therefore,in this study,we first designed a series of new experimental programs and technical means to explore the permeability of CCZ,aiming to uncover the permeability of CCZ in its developing and remodeling progress.Then,the molecular mechanism of CCZ development was further explored by dressing the expression and functions of VDCC in chondrocytes.MethodsPart I:Observation of the permeability in mature/immature CCZ of live mice in situ1.Histological observation of CCZ in mice of different ages.Twenty four Balb/c female mice(1-8 months old,3 in each month old)were sacrificed.The both knee joints were dissected,fixed,decalcifed,paraffin embedded and sliced.Then,Hematoxylin&Eosin(HE)staining and Saffron O/Fast green staining were performed.Image J software was used to calculate the thickness of cartilage(the distance from the cartilage surface to the tide mark)and the thickness of CCZ(the distance from the tide mark to the cement line)2.Design and manufacture of mouse fixation deviceL-type stainless steel plate,installed with two mechanical arms in proper position.One of the arms was fixed the femur of mouse leg,and the other one was fixed the self-manufactured plastic culture dish with hole.The femur of mouse knee joint was exposed and passed through the hole of the dish,and the surrounding area was closed with glue.3.The observation of the permeability of CCZ in vivo mice84 mice of 1 month and 6 months were divided into immature CCZ group(n=42)and mature CCZ group(n=42).The permeability of the mice was tested with Rhodamine B solution and Tetramethylrhodamine isothiocyanate dextran solution(TRITC-Dextran).Then the animals were divided into four subgroups:mature CCZ+rhodamine B,mature CCZ+TRITC-Dextran,immature CCZ+rhodamine B,immature CCZ+TRITC-Dextran.The diffusion time was 1 min,15 min,30 min,1 h and 2 h.The knee joint(0 min)without a penetration test was used as a blank control,while the distal femur was soaked in rhodamine B or TRITC-Dextran for 72 h as a positive control.4.Un-decalcified fluorescence observation technology and image analysisAfter knee joint samples were collected,the following treatment was carried out in sequence:freeze-drying dehydration,plastic embedding,cutting and grinding,confocal microscope scanning,DAPI and fluorescein sodium re-staining,confocal microscope re-scanning,and merging the images scanned in twice separately.Finally,the fluorescence value in different samples was quantitative analyzed and statistical test.Part II:Permeability of CCZ in human OA knee samples based on Micro-CT and Fick law1.Collection and processing of OA samplesAccording to the inclusion exclusion criteria,patients were selected.The samples were obtained after the total knee replacement operation.8mm osteochondral column(grade 0-1 or4)was drilled based on the surface morphology of cartilage.the osteochondral column was put into the centrifugal tube until UV resin glue submerge the sample but leave the cartilage surface untouched and solidified by UV.2.Mirco-CT scan and image analysisThe cartilage surface was added with 0.5 ml iodohydrin and immediately put into Mirco-CT for scanning.The first scan time was 0 h,and then scanned again at 6h,24h,48h and 72h.The image data are reconstructed by 3D slicer and Amira software,and the parameters of the samples are calculated.3.Diffusion coefficient calculation and mathematical modelingThe mathematical model is established according to Fick's law.The specific values are brought into the equation and fitted with experimental data to obtain the diffusion coefficient K in articular cartilage.Part?:The expression specificity of T-VDCC during the development of CCZ in mice.1.Tissue sections of knee joint of mice of each month.The paraffin blocks of knee joints of mice of each month(1-8 month)were routinely sliced.2.Immunohistochemical staining of mouse knee joint specimens.The sections of mouse knee joint samples at different developmental stages were immunohistochemical stained with T-VDCC and chondrocyte hypertrophy and ossification proteins,then observed and statistical analyzed.Part IV:Molecular mechanism of chondrocytes hypertrophic/osteogenic differentiation after Cav3.3 inhibited.1.Extraction and culture of primary mouse chondrocytes and ATDC5 chondrocyte line.Balb/C mice 7-10 days old were selected.Chondrocytes were isolated and cultured from articular cartilage after sacrificed.It was cultured and expanded in vitro under the same conditions as ATDC5 cell line.P2 generation mouse chondrocytes were used for experimental treatment and related detection.2.Expression of T-VDCC in mouse primary chondrocytes and ATDC5 chondrocyte line.Three subtypes of T-VDCC(Cav3.1,Cav3.2,Cav3.3)immunofluorescence staining and western blot were performed on two kinds of cells to explore the natural expression of these three subtypes in these two kinds of cells.3.Effects of different concentrations of Mibefradil on inhibition of T-VDCC on ATDC5chondrocytes.After adding 1?m,5?m,10?m and 15?m Mibefradil to culture medium of ATDC5chondrocytes 48 h,apoptosis detection by flow cytometry,Alcian blue,alizarin red,alkaline phosphatase staining,immunofluorescence staining and western blot were performed.The expression of Cav3.1,Cav3.2,Cav3.3 and protein markers of cartilage phenotype and hypertrophy phenotype were detected.4.The effect of si RNA on the expression of Cav3.3 in ATDC5 cells.According to the previous experimental results,ATDC5 cells were transfected Cav3.3-si RNA to inhibit the expression of Cav3.3.24 h later,RT-PCR,immunofluorescence staining and western blot were performed to detecte the expression of Cav3.1,Cav3.2,Cav3.3to confirm whether Cav3.3 was knocked down.72 h after transfection,apoptosis rate,Alcian blue,alizarin red,alkaline phosphatase staining,immunofluorescence staining and western blot were performed.Results:Part ?:Observation of the permeability in mature/immature CCZ of live mice in situ.1.The CCZ in knee joint of 6-month-old mice is mature.The knee joints of 1-5 month-old mice were immature.The tide mark was still not obvious,and the boundary of each structure was not clear.The knee joints of 6-month-old mice were basically mature,and the boundaries of cartilage,CCZ and subchondral bone were obvious,and the tide mark and cement line could be seen clearly.The joints of 7 and8-month-old mice were well developed and the subchondral bone became denser.2.The mature CCZ was able to block the diffusion of rhodamine B,while the immature CCZ did not.The time for the rhodamine B(476 Da)diffused in the cartilage layer of the mature CCZ group reaching the saturated concentration was 30 min and that is 1 h in immature CCZ group.After diffusing for 2 h,in the mature CCZ group,there was no fluorescence of rhodamine B below the tide mark,but it could be detected in the immature CCZ group.3.Mature CCZ was able to block the diffusion of TRITC-Dextran,but immature CCZ did not.For mature CCZ group and immature CCZ group,the time for TRITC-Dextran(20k Da)to reach saturation concentration in cartilage layer was 30 min and 1 h,respectively.After diffusing 2 h,there was a low concentration of TRITC-Dextran in the subchondral bone in the immature CCZ group,suggesting that TRITC-Dextran penetrated into the subchondral bone through the immature CCZ,while it could not be detected in the mature CCZ group.Part ?:Permeability of CCZ in human OA knee samples based on Micro-CT and Fick law.1.Compared with the normal CCZ,the structure of the CCZ of OA(grade 4)changed and the permeability increased.Micro-CT scan showed that the thickness and porosity of CCZ increased in OA(grade 4),compared with normal tissue(grade 0-1).After 72 h of infiltration,the CT value in subchondral bone of OA(grade 4)increased,implying that the iodide alcohol reached there,but the iodide alcohol in normal tissue subchondral bone was not obvious.2.Change of diffusion coefficient of CCZ during OA process.According to the data and mathematical model,the diffusion coefficient of normal CCZ is 0.03±0.01?m~2/s,while that of grade 4 OA CCZ is 0.12±0.04?m~2/s,which means that the solute exchange between cartilage and bone is easier in grade 4 OA.Part ?:The expression specificity of T-VDCC during the development of CCZ in mice.1.The expression of T-VDCC decreased gradually during the maturation of mouse CCZ.With the development of mouse knee joint,the tissue in deep layer(CCZ forming area)gradually calcified,and the expression of Cav3.1,Cav3.2,and Cav3.3 in chondrocytes decreased accordingly,accompanied by the high expression of COL 10 and osteocalcin in this region.However,Cav3.1,Cav3.2 and Cav3.3 continuously expressed in the surface and middle cartilage layers from 1 to 8-month-old.While the expressions of COL 10,osteocalcin and MMP 13 were only found at the age of 1 and 2 months in surface and middle cartilage layers,and then no longer expressed after that.Part ?:Molecular mechanism of chondrocytes hypertrophic/osteogenic differentiation after Cav3.3 was inhibited.1.Among three subtype T-VDCC,Cav3.3 was mainly expressed in primary mouse chondrocytes and ATDC5 cells.The immunofluorescence staining of Cav3.1,Cav3.2,and Cav3.3 of primary chondrocytes and ATDC5 cells indicated that the fluorescence of Cav3.3 is obvious in the both kinds of cells,while the fluorescence of Cav3.1 and Cav3.2 was almost invisible.The western blot of the three subtypes showed that Cav3.3 bands were clearly visible,while Cav3.1 and Cav3.2 had no bands.2.Mibefradil inhibits the expression of Cav3.3 in ATDC5 cells.When 1?m,5?m,10?m and 15?m Mibefradil were added to the culture medium,the PCR results showed that the expression of Cav3.1,Cav3.2 and Cav3.3 decreased with the increasing concentration,and the trend of Cav3.3 was the most obvious.Western blot showed the same results,but Cav3.1 and Cav3.2 also had no bands,and the expression of Cav3.3decreased most significantly at the concentration of 10?m,which was consistent with the result of immunofluorescence.3.Mibefradil promotes the apoptosis,the loss of cartilage phenotype and the tendency of hypertrophy and osteogenesis in ATDC5 cellsWhen 1?m,5?m,10?m and 15?m Mibefradil were added to the culture medium,the proportion of early apoptosis of ATDC5 cells increased with the increasing inhibitor concentration,Alcian blue staining became lighter,while Alizarin red and ALP osteogenic markers staining increased.Western blot suggested that with the increase of inhibitor concentration,the expression of ACAN,COL 2 and SOX 9 decreased,while the expression of COL 10,MMP 13 and RUNX2 increased,among which,5?m and 10?m were the most significant.When 10?m Mibefradil was added to the culture medium,the expression of COL2 and SOX 9 decreased,while the expression of COL 10,MMP 13 and RUNX2 increased by immunofluorescence staining.It is suggested that after the inhibition of T-VDCC,the phenotype of ATDC5 cartilage was lost and the osteogenic tendency appeared.4.After si RNA transfection into ATDC5 cells,the expression of Cav3.3 was down-regulated,the phenotype of cartilage changed,and there was a tendency of hypertrophy and osteogenesis.After successful transfection of constructed Cav3.3-si RNA,PRC,western bolt,and immunofluorescence staining indicated that the expression of Cav3.3 in ATDC5 cells was down-regulated.After transfection,the proportion of early apoptosis increased,and the staining of Alizarin blue,Alizarin red and ALP indicated the loss of cartilage phenotype,showing the tendency of osteogenesis.Immunofluorescence staining and western blot showed that the expression of COL 2 and SOX 9 decreased,while the expression of COL 10,MMP 13and RUNX2 increased.5.After inhibition of Cav3.3,chondrocytes show osteogenic tendency dependent on osteocalcin/NFAT pathway.After the down-regulation of Cav3.3 expression in ATDC5 cells,immunofluorescence and western blot showed that the expression of COL 1 and non-phosphorylated NFATc2increased significantly,while the expression of osteocalcin,phosphorylated NFATc2 increased slightly,suggesting that this osteogenic tendency is though Calcineurin/NFAT pathway.Conclusion:1.At the age of 6 months after birth,the calcified cartilage zone of the joint is mature,and the mature CCZ has a barrier function.2.During the remodeling of CCZ in severe OA,the increase of porosity and diffusion coefficient facilitated the solute exchange between articular cartilage and subchondral bone.3.With the calcification and maturation of CCZ in mice joint,the expression of T-VDCC in chondrocytes within CCZ decreased gradually,while the expression of hypertrophy and ossification marks increased.4.Cav3.3 was able to maintain cartilage phenotype.ATDC5 cells appeared hypertrophic and osteogenic differentiation tendency through calcineurin/NFAT signaling pathway after inhibiting the function and expression of Cav3.3.