Experimental Study Of Exogenous Decorin On The Extracellular Matrix Of Articular Cartilage Cells In Vitro

Background：Osteoarthritis （OA） is a kind of chronic degenerative disease causing joint disorderand is characterized by the destruction of articular cartilage, subchondral bone remodelingand synovitis, which is considered to be the most prevalent chronic joint disease, and itsincidence continues to rise due to the ageing of the population and the prevalence of obesity.The main clinical features are pain and loss of joint function. Current treatment is mainlyconcentrated in the remission of symptoms, including nonpharmacologic, pharmacologicand surgical methods, but these methods are not able to control the progression of thedisease. Articular cartilage belongs to hyaline cartilage, which has no blood supply, nolymph drainage and no innervation, and as a bearing material, it endures the impact forceand shear force. Chondrocyte is the only cell component of articular cartilage. These cellssynthesize and maintain the extracellular matrix, including collagen and proteoglycan.Collagen provides tensile strength of cartilage tissue, and proteoglycan, especially aggrecan,maintains a large amount of water molecules in the cartilage matrix, which lets the cartilagetissue withstand the pressure and load. Accumulating evidence suggests that cartilage losscaused by the imbalance of anabolism and catabolism of cartilage matrix and the enhancedcatabolism of extracellular matrix plays an important role in osteoarthritis.Decorin （DCN） is a small leucine-rich proteoglycan and belongs to the smallleucine-rich proteoglycan （SLRP） family, and it is the first small proteoglycan cloned fromhuman embryonic fibroblast cell line, which was known as PG40, and it was renameddecorin because it shows modification of collagen fibers in the electronic the microscope.DCN widely exists in the matrix of collagen I and II, and involves in important biologicalfunctions such as: structural fabric of matrix, regulation of cell adhesion, migration andproliferation. DCN combines with transforming growth factor beta1,2and3(TGF-β_1,2,3)through its core protein, and inhibits their activities. Studies have shown that the decomposition fragments of DCN increased in the degenerate articular cartilage. In earlyosteoarthritis, gene expression of DCN in articular cartilage was inhibited; while inadvanced osteoarthritis, the content of DCN in articular cartilage decreased andtranscription and translation of DCN raised. These results suggested that DCN wasimportant in the occurrence and development of osteoarthritis. With the method of primarycell culture in vitro, the experiment was to observe gene expression changes of cartilageextracellular matrix degrading proteases （MMP13, ADAMTS-5） caused by differentconcentrations of exogenous DCN and gene expression changes of chondrocytehypertrophy （Collagen X） to discuss its potential mechanism.Objective：To investigate the gene expression changes of extracellular degrading proteases（MMP13, ADAMTS-5） of chondrocytes and hypertrophy （Collagen X） of chondrocytes onthe impact of exogenous DCN with different concentration，through culturing primarychondrocyte from normal human articular cartilage in vitro.Methods：1. Specimen sourceFive specimens of full-thickness articular cartilage were resected in artificial total hiparthroplasty of femoral neck fracture, and the ages were ranging from63to87（77.2±9.01）,including2male cases and3female cases. The obtainment of specimens was approved bythe hospital ethics committee, and informed and agreed by the patients.2. Isolation and culture of human articular cartilage cellsThe isolation of articular cartilage cells was referring to the method of monolayerculture. Articular cartilage tissues obtained from the above patients were rinsed severaltimes by1x PBS under aseptic conditions, and excess tissue surrounding articular cartilagewere removed after the digestion for15min by2.5g/L trypsin. After being rinsed by1xPBS, articular cartilage tissues were sheared into fragments of1mm3, and digested with0.2%collagenase II （Gibco） containing10%fetal calf serum for15-20h, after the digestion,the cell suspension was filtered with150mesh stainless steel mesh, and then centrifuged for5min in1000R/min and centrifuged again after being rinsed by1xPBS. The cells werecounted by trypan bule staining and inoculated in12-well plates with proper DMEM/F12（containing10%fetal bovine serum,100IU/ml penicillin and100μg/ml streptomycin） with the cell density of5.5x10⁵/hole, and were cultured in the CO2culture box （37℃, saturatedhumidity and5%CO₂）. Culture solution may be replaced after24h according to cellattachment, later be replaced every two or three days. The growth of cells was observed byinverted microscope. When the cell fusion reached more than80%（on the fifth day）, theplates were added exogenous DCN （Sigma） solution to reach the concentrations of0,2.5,25,250μg/ml to be used after24h.3. Identification of human articular chondrocytes cultured in vitro3.1Alcian blue stainingPrimary chondrocytes cultured for5days were washed for2times with PBS, and fixedfor20min in4%paraformaldehyde, and then washed for2times with PBS and stained for15min in1%alcian blue, and then rinsed with distilled water.3.2Immunohistochemistry of collagen IIPrimary chondrocytes after the separation, purification and counting were inoculatedon the coverslips placed in the12-well plates, and were removed from the plates afterculturing for5days, and then fixed with the method same as alcian blue staining. Theywere blocked with normal goat serum for20min and were added dropwise of collagen IImonoclonal antibody （Sigma） diluted with1:50, and incubated overnight at4℃. Andbiotinglated secondary antibodies were added to incubate for1h at room temperature.SABC reagent was added to incubate for20min at room temperature. DAB reagent wasadded to stain, and the dyeing time was controlled in the microscope, and then dehydratedand mounted.4.RNA extraction and reverse transcription of primary cartilage cellsIn the conditions with the absence of RNA enzyme pollution, RNA primary cartilagecells on the sixth day were extracted using Trizol, following by reverse transcriptionaccording to Takara kit instruction.5. Real time PCRIt was performed in MX3000P PCR tester, and Real time PCR reaction reagent kit wasfrom TaKaRa （SYBR TM Premix Ex TaqTMKit, Japan）. Detect mRNA expression levels oftarget genes from the control group and experimental groups: Collagen X, MMP13andADMTS-5. The preparation of PCR reaction system and the setting of amplificationconditions referred to quantitative PCR reagent kit instruction, and there were3parallel holes for all reactions. PCR instrument automatically provided melting curve analysis to getthe amplification curves, melting curve and sample cycle threshold （Ct） value. Internalcontrol gene was glyceraldehyde-3-phosphate dehydrogenase （GAPDH）.6. Statistical methodsData were expressed as x±S, and independent t-test was performed with SPSS10.0program. A p value of≤0.01was considered significant.Results:1. Morphological observationPrimary cartilage cells just isolated have well-distributed size and were spherical withhighly refracted. Primary cartilage cells would generally adhere in24h, and primarychondrocytes after adhering were flat with multi angles and abundant cytoplasm, withround nuclei in the centre and2to3clearly visible nucleolus, and there may be colonelgrowth in some regions. Primary chondrocytes cultured for5days after replacing the liquidfor2³times were polygonal or star and were closely arranged “slate”, while the cellswere differentiated after the seventh day, and the cells gradually evolved to shuttle, with aplurality of finger-like protrusions and fibroblastic morphology.2.Alcian blue staining and immunohistochemistry of collagen IIGlycosaminoglycan （GAG） and collagen II are important components of hyalinecartilage extracellular matrix, which can be used as the main indexes of functionalidentification of hyaline cartilage cells cultured in vitro. The experiment stained primarycartilage cells cultured in vitro with the methods of alcian blue staining and collagen IIimmunohistochemistry, and found it positive. Normal chondrocytes in monolayer growthzone and multilayer growth region were stained to be pale blue by alcian blue staining, andthe extracellular matrix at the center portion of colony growth region and multilayer growthregion were blue, and the immunostaining of collagen II was thick brown, being positive,which confirmed their properties of hyaline cartilage cell.3.mRNA expression levels of related genes in primary cartilage cells detected by Realtime PCRWith the increasing concentrations of exogenous DCN （0,2.5,25,250μg/ml）, theexpression of ADAMTS-5and Collagen X were significantly reduced （P<0.01） with adose-dependent manner, while the expression of MMP13was significantly lower in high concentration group of DCN （250μg/ml）（P <0.01）.Conclusion:1.Our study demonstrates hyaline cartilage cell morphology and functional propertiesof the monolayer culture method in cultured normal human primary articular chondrocyteswithin five days, seven days after the gradual emergence of dedifferentiation.2.Our study demonstrates that exogenous DCN inhibition of chondrocyte hypertrophy,inhibition of the expression in the cartilage cells to extracellular matrix degradingenzymes（MMP13,adamts-5）.