The Study Of Mechanism On Mechanical Loading Regulating Chondrocyte Phenotype

Background:Osteoarthritis（OA）is a joint degenerative disease that is significantly related to mechanical loading,cartilage degradation and matrix loss is the leading pathological characteristics in OA.As the major cell type in cartilage,chondrocytes have an important role in maintaining the healthy status of cartilage by keeping their extracellular matrix anabolic and catabolic balance.At present,the mechanism on mechanical loading regulating chondrocyte phenotype still needs to be further studied and elucidated.Objective:The study aims to explore upstream regulators that could not only function as a mechano-transductive effector in chondrocytes,but also control cartilage phenotype and participate in OA pathology.Comparing the differentially expressed genes（DEGs）of preserved cartilage and damaged cartilage from OA knee joint through transcriptome sequencing;Predicting the upstream molecules that could regulating DEGs,verifying its potential of conducting mechanical loading signals and influencing chondrocytes phenotype.Methods:1.Obtaining cartilage tissue from the advanced osteoarthritic knee joint of patients underwent total knee joint arthroplasty（TKA）,Outerbridge score and OARSI score were used to grade the cartilage from the macroappearance and histopathological level to distinguish the preserved cartilage area（P-C）and severely damaged cartilage area（D-C）.2.Immunohistochemistry staining was used to detect the expression of cartilage degradation and chondrocyte senescence-related proteins;P-C and D-C were digested with type 2 collagenase to obtain primary chondrocytes（P-CH,D-CH）,qPCR and Western blot method to detect the gene and protein expression level of the osteoarthritis related markers;After 14 days of in vitro chondrocyte three-dimensional pellet culture,Safranin O/Fast green staining detects the synthesis of hyaline cartilage matrix,and qPCR and western blot detects the gene expression level of cartilage and osteoarthritis related markers.3.The primary P-CH and D-CH were lysed and total RNA was extracted using Qiazol solution.Transcriptome analysis was performed by high-throughput RNA-sequencing method.R,cytoscape,metascape software were used for identifying significant differentially expressed genes（DEGs）between P-CH and D-CH,and ingenuity pathway analysis（IPA）was used to predicting upstream transcriptional regulators that can be used to explain observed DEGs.4.qPCR,immunohistochemistry staining and western blot were used to measure gene and protein expression level of ESR1（ERα）in P-C and D-C from TKA patients.Through acquiring and downloading other two transcriptome data from GEO,we conducted the same analytical pipline to verify the ESR1 gene expression level in other studies.Meanwhile,immunohistochemistry staining evaluated ERαprotein level in articular cartilage from 3-month C57BL/6 WT mice,24-month C57BL/6WT mice and 24-month OA mice.5.ESR1-overexpression lentiviruses and lipofectamine containing siRNA of ESR1 were used in P-CH and D-CH,protein expression level of chondrogenic markers,proliferative markers,senescent markers and osteoarthritic markers were evaluated by western blot.Meanwhile,in order to verify the effects of ERαin maintaining healthy status of articular cartilage,we established esr1 gene knock out mice(esr1^-/-)and compared articular cartilage status with their wide type littermates through pathological staining and OARSI score.6.15%methacrylated gelatin（Gel MA）mixed with 10 million/m L P-CHs was photo-crosslinked to a solid status and applied under mechanical loading stimulation to mimic knee joint movement.qPCR,immunofluorescent staining and western blot were used to evaluate gene and protein expression level of ESR1（ERα）,cartilage degradation markers and cellular senescent markers were detected as well under different loading situation.7.ESR1 gene expression was first silenced in P-CH by siRNA transfection,and then siESR1-P-CH were encapsulated in Gel MA and submitted under different mechanical loading.qPCR,immunofluorescent staining and western blot were used to evaluate the change of gene and protein expression level after removing ERαin chondrocytes;Immunofluorescent staining was applied to test the protein expression level of osteoarthritic markers in C57BL/6 WT and KOesr1 mice.Results:1.According to Outerbridge score,P-C was preserved cartilage area with Outerbridge score<1,D-C was severely damaged area with Outerbridge score>2;OARSI score evaluated histology slice stained by Safranin O/Fast green solution,OARSI score of all P-C area<10,OARSI score of all D-C area>18.Intra-and interobserver agreement was analysed based on the intraclass correlation coefficient（ICC）,with the parameter greater than 0.8 was considered to have good agreement.2.Safranin O/Fast green staining revealed that D-C had severe cartilage degeneration level and GAG loss.Immunohistochemistry staining and western blot showed that compared with P-CH,D-CH had higher protein expression level of cartilage degeneration marker（MMP13）and chondrocyte senescent markers(p16^INK4a,p21),and also exhibited stronger senescence-associatedβ-galactosidase（SA-β-gal）positive staining.qPCR results showed in D-CH,the expression level of ADAMTS4increased by 2.437±1.218 times（P<0.0001）,ADAMTS5 increased by1.878±0.672 times（P=0.0006）,MMP13 increased by 2.519±1.184 times（P<0.0001）,and the expression of senescence-related gene p16^INK4aincreased by 2.793±1.727 times（P<0.0001）,the expression level of inflammation-related gene IL8 increased by 5.184±6.678 times（P=0.001）,the expression of fibrosis-related gene COL1 increased by 4.969±4.036times（P<0.0001）,and the expression of COL3 increased by 2.899±2.121times（P=0.0092）,the expression of hypertrophy-related gene RUNX2increased by 1.884±0.826 times（P<0.0001）,and the expression of ossification-related gene OPN increased by 24.790±20.032 times（P<0.0001）;qPCR and western bolt showing that in vitro three-dimentional cultivation of D-CH still maintained their high senescent level and osteoarthritic level.3.Bulk RNA-sequencing results revealed that compared with primary P-CH,there are 313 up-regulated genes and 245 down-regulated genes in D-CH;Gene ontology enrichment analysis showed DEGs majorly enriched in extracellular matrix region,receptor and ligand activity,cell adhesion molecule binding function etc.;KEGG（Kyoto Encyclopedia of Genes and Genomes）and Reactome pathway enrichment analysis showed that extracellular matrix degradation pathway,assembly of collagen fibrils and other multimeric structure pathway,regulation of insulin-like growth factor（IGF）transport and uptake pathway,cellular proliferative related PI3K-Akt pathway etc.were the most significant enriched pathways.The results of predicted up-stream regulators by IPA suggested that NEUROG1（p-value of overlap=1.65E-10）,estrogen receptor（p-value of overlap=2.26E-10）,HNRNPA2B1（p-value of overlap=2.71E-10）,TGFB1（p-value of overlap=1.74E-08）,ESR1（p-value of overlap=1.85E-08）are the top 5 important molecules that can regulate DEGs change;ESR1belongs to the estrogen receptor family,and its downstream target genes include IGF1,IGFBP2,IL6,MMP1,RUNX2,BMP2,CDKN2A and other OA-related genes.4.Immunohistochemistry staining and western blot results confirmed that compared with P-C（P-CH）,D-C（D-CH）had lower ERαprotein level,and the expression level of ERαdid not correlate with gender.qPCR results indicated that gene expression level of ESR1 was significantly decreased in primary D-CH from other 6 OA patients underwent TKA（P<0.05）;The results of analysing the other two transcriptome data（E-MTAB-4304,GSE57218）from GEO showed that ESR1 gene expression level was the highest in healthy cartilage and the lowest in severe damaged cartilage.Immunohistochemistry staining revealed that the protein expression level of ERαwas significantly decreased in C57BL/6 aged and OA mice,when compared with 3-month WT littermate control.5.Western blot results showed that after eliminating ESR1 in P-CH（siESR1 P-CH）,the protein level of ERα,SOX9 were decreased,ADAMTS4,MMP13,p16^INK4a were increased;After transfecting ESR1activation lentivirus into siESR1 P-CH to recover the expression of ESR1,western blot results revealed that the expression of SOX9 was enhanced and the expression of ADAMTS4,MMP13,p16^INK4a were inhibited;Meanwhile,ESR1 activation lentivirus was also transfected into primary D-CH,western blot results found that the protein level of ERαcould be partially reversed,and the down-stream targets including COL2A1 protein level was increased too,while the protein expression level of ADAMTS4,MMP13,p16^INK4a were significantly inhibited.Through comparing knee joint sectioning and pathological staining results of 3-month-old esr1^-/-mice and wild type littermate control,we found that the safranin O positive staining rate was extremely lower than control groups,even though there were no obvious cartilage degradation and vertical fissures in esr1^-/-mice.The semi-quantification of the loss of proteoglycan grade showed that wide type littermate control was 0.125±0.250 and esr1^-/-mice was 3.250±1.190,P value equals to 0.0177.6.qPCR results showed that compared with non-mechanical loading stimulated P-CH,20%loading strain caused gene expression level of ESR1decreased by 0.185±0.088 times（P=0.0003）,and its downstream MMP13gene expression level increased by 4.920±1.163 times（P<0.0001）,p16^INK4aincreased by 3.390±0.352 times（P<0.0001）,p53 increased by 2.318±0.296times（P=0.0160）,OSX increased by 2.337±0.455 times（P=0.0257）,VCAN increased by 4.960±0.648 times（P<0.0001）;The results of immunofluorescent staining and western blotting also verified the change trend at the protein expression level.Western blot also showed that the expression level of ADAMTS4,inflammation-related protein pho-p65were increased under the stimulation of mechanical loading.7.Comparing with non-mechanical loading stimulated siESR1 P-CH,the gene expression level of siESR1 P-CH under 20%loading strain had dramatically changed.ADAMTS4（siESR1 control:3.786±0.386,siESR1 20%loading:2.364±0.148,P=0.0287）,MMP13（siESR1 control:4.870±0.043,siESR1 20%loading:2.396±0.780,P<0.0001）,p16INK4a（siESR1 control:5.284±0.770,siESR1 20%loading:1.639±0.343,P<0.0001）,these three genes expression level decreased when ESR1expression was depleted;however,the genes related to cartilage hypertrophy,fibrosis and cartilage ossification were up regulated,like COL10（siESR1 control:1.957±0.027,siESR1 20%loading:4.349±0.507,P<0.0001）,OCN（siESR1 control:2.254±0.143,Si ESR1 20%loading:5.628±0.366,P<0.0001）,OPN（siESR1 control:0.888±0.250,siESR1 20%loading:2.239±0.494,P=0.0238）,OSX（siESR1 control:0.881±0.210,siESR1 20%loading:3.473±1.422,P<0.0001）,VCAN（siESR1 control:0.817±0.337,siESR1 20%loading:2.036±0.531,P=0.0127）;The high protein expression level of COL10 and OCN in siESR1 P-CH under 20%loading strain were approved through immunofluorescent staining and western blot.Besides that,immune-staining showed that the protein level of OCN was highly expressed in D-C compared with P-C（P-C:87.555±11.517,D-C:132.800±13.319,P=0.0024）,and also in articular cartilage from C57BL/6 KOesr1 mice compared with C57BL/6 WT mice（WT:3.156±2.022,KOesr1:17.679±8.046,P=0.0128）.Conclusions:1.Due to the different distribution of mechanical loading within knee joint,D-C had severe osteoarthritic pathological changes and chondrocyte senescent level compared with P-C collected from the same knee joint.2.Upstream regulator ERαplays a role in maintaining healthy cartilage phenotype,manipulating senescent level and sensing mechanical loading in chondrocytes.3.Supraphysiological compressive strains（20%）could inhibit ERαexpression level and further induce cartilage degeneration and senescent related phenotype.4.Depleting ERαcould dramatically change the response of P-CH to 20%loading strain,cause chondrocytes hypertrophy and ossification.