The Effects And Mechanism Of Paracrine Proteins Derived From Mesenchymal Stem Cells In Promoting Cartilage Repair

Articular cartilage lesion is one of the most common joint diseases in clinic.Because of no vascular supply,no innervation and no lymphatic circulation,plus poor migration and proliferation ability of the chondrocytes in the cartilage tissue,different kinds of injury as well as inflammation and degeneration can cause irreversible cartilage damage.At present,available drugs are mainly used to relieve pain as a conservative treatment,which can't achieve prefect cartilage repair.Though doctors of joint surgery are actively trying to explore a better way of treatment,the current microfracture,autologous cartilage implantation(ACI)and other technologies are still insufficient to achieve perfect regeneration of cartilage with normal structure and function.Owning to the wide sources,low immunogenicity and multi-directional differentiation property of stem cells,the cartilage tissue engineering based on stem cells has become a new therapeutic strategy for cartilage regeneration.With the growing evidence demonstrating that the paracrine of mesenchymal stem cells(MSCs)plays an important role in promoting cartilage repair,the MSCs secretome based cell-free therapy has attracted more and more attention.In order to discover the underlying mechanism of MSCs secretome in cartilage repair,we conducted transmission electron microscopy(TEM)characterization,cytokine microarray test,proteomics and bioinformatics analysis on the MSCs secretome in this study.Finally,we have revealed the role of MSCs derived soluble proteins(mainly RCN2)played in restoring cartilage matrix and elucidated the possible mechanism,which could provide an alternative for the treatment of articular cartilage defects.The main results are as follows:1.In the presence of IL-1 ? when culturing rat chondrocytes in vitro,the adding of rat BMSCs derived soluble proteins(SPs)could better help maintain the normal morphology and phenotype of chondrocytes.While the exosomes(Exos)group and micro-vehicles(MVs)group presented no difference,suggesting that MSCs secretome protect the morphology and phenotype of chondrocytes better under the influence of IL-1 ? is not attributed to Exos or MVs but the SPs,which is further confirmed in the OA rat model in vivo.2.Comparing CM derived from ADSCs/ BMSCs with different 2D/3D culturing conditions,PCR and Western blot tests showed that CM derived from ADSCs was more conducive to maintaining chondrocytes' chondrogenic phenotype than BMSCs';and the CM collect from 3D culture was better than that from 2D when comparing2D/3D ADSCs groups,which means the change of the diameter of 3D scaffolds had little effect on the ability of CM in maintaining chondrocytes' chondrogenic phenotype.Proteomics analyze found that the number of differential protein of SPs between 2D/3D ADSCs groups was significantly higher than that between 3D groups with different scaffold pore sizes,indicating that the difference of 3D scaffolds' diameter had little influence on the expression of SPs in the CM.Through further bioinformatics analysis,we selected four molecules most likely to influence the phenotype of chondrocytes from SPs derived from ADSCs: CAPNS1,LAL,MAT2 A and RCN2.sh RNA transfection test of the four molecular showed that after down-regulation of RCN2: the expression of MMP-13 significantly reduced,and the expression of SOX-9 increased,barely no influence on the expression of Col-X and Col-I,suggesting RCN2 may be the main functional protein in the SPs in regulating chondrocyte chondrogenic phenotype.3.To investigate the mechanism how RCN2 regulates chondrocytes' chondrogenic phenotype,we established stable human/rat chondrocyte RCN2down-regulated and up-regulated cell lines.Results showed that the expression of matrix-degrading enzymes MMP-13 and ADAMTS-5 in chondrocytes decreased after knocking-down RCN2,and cartilage matrix protein Col-II,SOX-9,ACAN increased.While,after up-regulation of RCN2 the expression of matrix-degrading enzymes MMP-13 and ADAMTS-5 increased,and cartilage matrix protein Col-II,SOX-9,ACAN decreased.Further experiments found RCN2 down-regulation lead to NFAT1,NFAT2 expression increase,while up-regulation on the contrary.Suggesting that RCN2 may regulate chondrocytes' synthesis of cartilage matrix through NFAT signaling pathway.In conclusion,this study confirmed that SPs in the CM of BMSCs could better help maintain the morphology and chondrogenic phenotype of chondrocytes in the presence of pro-inflammatory cytokine IL-1? than EXOS and MVS.CM derived from ADSCs was superior to BMSCs in cartilage repair,and CM obtained from 3D scaffolds culture was superior to 2D culture,while changes in the pore size of 3D scaffolds had little influence on the function of CM.Proteomic analyze was conducted to screen out that RCN2 among SPs derived from ADSCs may affect the production of cartilage matrix,further experiments confirmed that RCN2 may regulate chondrocytes' synthesis of cartilage matrix through NFAT signaling pathway.