Effect And Mechanism Of Dynamic Mechanical Loading On Chondrocyte Phenotype And Drug Exploration

BackgroundOsteoarthritis(OA)is a chronic degenerative joint disease with high prevalence and high disability rate.At present,the pathogenesis of OA is believed to be related to long-term mechanical wear.However,due to the lack of ideal dynamic mechanical loading models,the role and mechanism of dynamic mechanical stress in the occurrence and progression of OA remains unclear,which limits the development of OA drug therapy.Therefore,constructing a reliable dynamic mechanical loading culture system is of great significance to promote the understanding of OA pathogenesis and develop disease-modifying OA drugs(DMOADs).PurposeIn this study,we aimed to construct a 3D cell-loaded hydrogel dynamic loading culture system to model moderate and excessive mechanical stress on human chondrocytes regarding chondrogenesis,inflammation and cellular senescence.Using this system as a platform,we investigated the molecular mechanisms and regulating pathways of mechanical loading on chondrocyte phenotype and evaluated a potential DMOAD,urolithin A,targeting loading impair.Method1.Human chondrocytes were harvested from the knee joints of deidentified donors without joint disease.Isolated chondrocytes were cultured,detached,and passaged.Passage 2(P2)chondrocytes were mixed with 2% hyaluronic acid and photo-crosslinked to get cylindrical cellloaded hydrogels for mechanical loading testing.Samples were divided into 3 groups,control group(with no compression),5% strain group and25% strain group.The Mechano-Active Transduction and Evaluation(MATE)bioreactor was used to apply periodic dynamic mechanical stress with the frequency of 1 Hz,3600 cycles per day for 14 days.qRT-PCR and elastic modulus test were applied to evaluate the properties of gels.2.2% hyaluronic acid was used for the scaffold of cell-loaded gels.MATE was used to apply periodic dynamic mechanical stress of different intensities.After 14 days,the static culture group(Static),the low-intensity mechanical stress(DL-5%)group and the high-intensity mechanical stress(DL-25%)group samples were collected for analysis.qRT-PCR was used to detect the expression of chondrogenesis-related genes(SOX9,COL2,ACAN),matrix degradation-related genes(MMP12,MMP13,ADAMTS5),inflammation-related genes(NF-κB,IL-6,IL-8),and cellular senescencerelated genes(CDKN1A,CDKN2 A,TP53).Safranin O-Fast green staining was used to detect the content of extracellular matrix(ECM)proteoglycan(GAG).COL2 protein immunohistochemical(IHC)staining was used to detect the content of type Ⅱ collagen.β-galactosidase(SA-β-Gal)staining was used to detect intracellular β-galactosidase activity.p21 and NF-κB protein levels were detected by IHC staining.3.Western blotting(WB)was applied to analyze the content of chondrogenic proteins(COL2,ACAN,SOX9),aging related proteins(p16,p21,p53),matrix degradation protein(MMP13),inflammation related protein(NF-κB),autophagy-related proteins(LC3B,LAMP1),apoptosis pathway-related proteins(BAX,CASP3),mitochondrial function-related proteins(PINK1,Parkin),oxidative stress-related proteins(NRF2)and mitophagy suppression regulator(p-ERK1/2)in different groups.Image-J was used to analyze the grayscale of the protein bands.Mitochondrial membrane potential was verified by fluorescence(JC-1)staining.4.Different concentrations(0,1 μM,10 μM,100 μM)of urolithin A(UA)were added into the culture medium of cell-loaded 2% HA gels.qRTPCR and WB were applied to evaluate the gene and protein expressions of different groups.Proper concentration of UA was selected for next step.Mechanical injury-induced OA models were established using MATE systems with 25% dynamic compressive loading.Chosen concentration of UA was added to the culture medium of the intervention group.qRT-PCR and WB were used to detect and compare the expression levels of genes and proteins mentioned above.Safranin O-fast green staining was used to detect the GAG content in ECM.Immunofluorescence(IF)staining was used to detect the TOM20 protein expression level.IHC staining was used to detect the protein expression levels of COL2,p16,LAMP1 and NRF2.Results1.The 2% photo-crosslinked cell-loaded hyaluronic acid hydrogel maintained a relatively stable elastic modulus under long-term culture and had no obvious influence on the phenotype of chondrocytes.It can be used as a 3D culture scaffold for studying the effect of dynamic mechanical stress on chondrocytes.2.Compared with Static group,DL-5% group showed significantly increased expression of COL2 gene.Differently,DL-25% group showed a significant decrease of COL2 and ACAN expression.Matrix degradationrelated genes MMP1,MMP13,ADAMTS5,inflammation-related genes IL-6,IL-8,and the expression of aging-related gene CDKN1 A were significantly higher in DL-25% than those in the Static group.The staining results showed that compared with the Static group,the staining intensity of GAG and COL2 in DL-5% group significantly increased but decreased in the DL-25% group.The IHC staining intensity of p21 and NF-κB and β-galactosidase staining significantly increased in DL-25% group.3.Compared with the Static group,the expression level of PINK1 in the DL-5% group was significantly increased.Expressions of p53,NF-κB and p-ERK1/2 were significantly decreased in the DL-5% group.In the DL-25% group,ACAN,LAMP1 and LC3 B were significantly decreased compared with the Static group.p21,p-ERK1/2 and PARK2 levels were significantly increased in DL-25% group compared with the Static group.JC-1 mitochondrial staining results showed that compared with the Static group,the staining intensity of JC-1 in the DL-5% group did not change much but decreased significantly in the DL-25% group.4.qRT-PCR results showed that compared with unmedicated group,10 μM UA can effectively inhibit the expressions of IL-6,IL-8,NF-κB and ADAMTS5 without effecting SOX9,ACAN,COL2,CDKN1 A and CDKN2 A.In the mechanical injury model,qRT-PCR results showed that compared with the injury group without UA(CON),the expression of ACAN and COL2 were significantly up-regulated in cells with 10 μM UA(UA group).While expressions of IL-6,IL-8,CDKN1 A,CDKN2A and ADAMTS5,MMP13 were significantly decreased.The staining results showed that compared with the CON group,the staining intensity of GAG and COL2 in UA group were significantly increased,while the staining intensity of p16 protein decreased dramatically.WB results showed that compared with the CON group,COL2,ACAN,SOX9,NFR2,LC3 B,LAMP1and TOM20 significantly increased in UA group.p-ERK1/2 protein was decreased in UA group.IHC staining showed that compared with the CON group,the staining intensity of LAMP1 and NRF2 in UA group was increased.IF staining result showed that the fluorescence staining intensity of TOM20 protein of UA group was higher than that of CON group.Conclusions1.Our self-constructed chondrocyte dynamic mechanical stress culture system based on 2% hyaluronic acid had stable mechanical and physical properties for testing and can be used for chondrocyte culture.2.Moderate mechanical loading had chondro-protective effects and promoted chondrogenesis.Excessive mechanical loading had detrimental influence on chondrogenic capacity,inducing inflammatory response,and stimulating chondrocytes to present an aging phenotype.3.Changes in mitochondrial function of chondrocytes may be an important mechanism for mechanical leading-induced chondrocyte degradation and OA progression.4.Urolithin A played a protective role against excessive mechanical loading-induced damage,probably by improving mitochondrial function in chondrocytes.