Screening Of Chondrocytes-derived Critical Soluble Factors Responsible For Chondrogenesis Of Bone Marrow Mesenchymal Stem Cells In Vitro And Optimization Of Strategies For Chondrogenic Induction

Background Damaged cartilage has a limited ability for self-repair, and has the tendency to develop into degenerative diseases if left untreated. Cartilage tissue engineering is emerging as a technique for the regeneration of cartilage tissue damaged due to disease or trauma. Among the various cell types that have been considered for cartilage tissue engineering, chondrocyte has been the most widely used cell source for its confirmative results of better engineered cartilage. But chondrocyte has its own shortages such as limited capability of proliferation, difficulties of repeatability and inferior mechanical properties. Bone marrow mesenchymal stem cell (BMSC), due to its easy way of harvest, multipotential differentiation capability and strong capability of expansion, has gradually become one of the optimal seed cell sources for cartilage engineering. But the induction methods used nowadays have shortages of low inducing efficiency, unstable results and nonphysical induction, all of which slow down the progressing of the application of BMSCs in cartilage tissue engineering. So it is imperative to develop efficient strategies for inducing chondrogenic differentiation of BMSCs.Studies have found that articular microenvironment can promote chondrogenesis of BMSCs. Chondrocyte is the only cell type in cartilage tissue and is one of the main components in articular microenvironment. In our previous study, we established an indirect co-culture system and found that soluble factors secreted from chondrocytes-scaffold constructs could induce chondrogenic differentiation of BMSCs via the trans-well. It is of important meaning to screen these critical inducing soluble factors secreted by chondrocytes-scaffold constructs for establishing a more efficient, induction strategy.However, soluble factors are too complex to be analyzed one by one. It is reasonable to find a high throughput screening model. Proteomics is the large-scale study of proteins with advantage of high throughput, and can be used to investigate complex changes of multiple factors and proteins. Protein microarray technology is another new technology raised in recent years in proteomics research with advantages of high throughput, high accuracy and sensitivity. It can provide considerable information in one experiment and enables us to study proteins comprehensively and accurately. The two technologies have gradually become important methods in research of stem cells and tissue engineering. So we plan to screen critical factors secreted by chondrocytes with capability of chondrogenic induction using technologies of differential proteomics and protein microarray.The first problem faced in the application of protein microarray and differential proteomics is selection of proper comparing targets. We hypothesize that the process of the chondrocytes-scaffold constructs gradually forming an engineered cartilage in vitro was similar to that of the cartilage development in vivo to some extend. Therefore, the cytokines and growth factors secreted from the engineered cartilage of early stage might be more in accordance with those needed in the chondrogenesis. But factors secreted from the engineered cartilage after long-term culture might be responsible for maintaining morphology of cartilage but not for chondrogenesis. So firstly we plan to compare the effects of chondrogenic induction of BMSCs between early-stage chondrocytes-scaffold constructs and late-stage chondrocytes-scaffold constructs. We want to prove that the supernatants of early-stage chondrocytes-scaffold constructs and late-stage chondrocytes-scaffold constructs can be the proper comparing targets for analysis of protein microarray and differential proteomics.Another essential problem faced in application of protein microarray and differential proteomics is the establishment of serum-free culture medium. High abundant proteins in serum will interfere with the analysis of results. What is more, in order not to interfere with the secretion of chondrocytes, the serum-free medium also cannot be added growth factors. Thereby we enrolled and investigated the effects of Insulin-Transferrin-Selenium (ITS), dexamethasone and vitamin C on chondrocytes proliferation, survival, secretion of extracellular matrix and formation of engineered cartilage to find an optimal combination of the hormones and chemical factors for establishing the serum-free medium used for engineered cartilage culture.On the basis of resolution of above mentioned two problems, we screened and compared the critical soluble factors secreted by early-stage chondrocytes-scaffold constructs and late-stage chondrocytes-scaffold constructs in the subsequent studies. Differential proteins were determined and their chondrogenic induction effects were tested. We hope to establish a clearly defined, stable and effective chondrogenic induction scheme and provide theoretical and technical support for the future clinical application of cartilage tissue engineering.Objection To screen the critical soluble factors secreted by chondrocytes-scaffold constructs which are responsible for chondrogenesis of BMSCs by means of protein microarray and differential proteomics, and to establish a clearly defined, stable and effective induction strategy for chondrogenic induction of BMSCs.Methods and Results1 Comparing the chondrogenic induction effects of soluble factors secreted by early-stage chondrocytes-scaffold constructs and late-stage cultured chondrocytes-scaffold constructsMethods BMSCs and mature chondrocytes were seeded onto biodegradable scaffolds respectively. Then the chondrocytes-scaffold constructs cultured for 3 days (3d group), 2 weeks (2w group) and for 8 weeks (8w group) were indirectly co-cultured with BMSCs-scaffold constructs respectively in a tans-well system to investigate the differential inductive effects of the soluble factors in all groups.Results BMSCs-scaffold constructs in 3d group developed into relatively uniform cartilaginous tissue with typical cartilage lacuna. BMSCs-scaffold constructs in 2w group only formed cartilaginous tissue at the rim, but fibrous tissue at the centre. BMSCs-scaffold constructs in 8 week group shrank generally and only formed fibrous tissue.Conclusion Soluble factors from chondrocytes-scaffold constructs at early stage had stronger directional effects of chondrogenesis than ones from chondrocytes-scaffold constructs at late stage.2 The establishment of serum-free mediumMethods P2 Chondrocytes were cultured in monolayer or in 3-D pellets in DMEM with 10% FBS (serum-containing group), and DMEM with 1%ITS,10-7M Dexamethasone, 50ug/ml Vitamin C (serum-free group). Chondrocytes proliferation, matrix secretion and gene expression were tested by MTT assay, histological staining, immunohistochemistry, and quantitative or semi-quantitative gene expression analysis.Results Serum-free culture medium could not promote chondrocytes proliferation in monolayer culture, while phenotype of chondrocytes could be maintained for at least 3 days. In 3-D culture, the histological, immunohistochemical results and expression of cartilage-specific genes were comparable between serum-containing group and serum-free group.Conclusion:Serum-free culture medium could promote cartilage formation and maintain phenotype of chondrocytes in 3-D culture. Serum-free culture medium can be used for differential proteomics analysis and protein assay analysis.3 Screening of critical soluble factors secreted by chondrocyte with capability of chondrogenic inductionMethods P1 chondrocytes derived from human costal cartilage were embedded in 1.2% Alginate gel and were seeded into PGA/PLA scaffolds. At designed time points, supernatants from serum-free media were harvested for testing. Supernatants before 2 weeks were regarded as early-stage samples and supernatants after 8 weeks were regarded as late-stage samples. Technologies of protein microarray and differential proteomics were used to screen critical soluble factors that may have chondrogenic induction effects.Results Differential proteomics found that 52 proteins were at least 2 times stronger in supernatants of early-stage than ones of late-stage.30 of the 52 different proteins were tested by mass spectrometry and 13 proteins were defined. Protein microarray found 6 growth factors secreted mainly at early-stage of chondrocytes-scaffold constructs, including IGF Binding Protein 2,4,6 (IGFBP2,4,6) and Fibroblast Growth Factor 2,6 (FGF2,6) and amphiregulin. IGFBP4 and IGFBP6 were selected as candidate factors and were proved to be expressed strongly at early stage by chondrocytes-scaffold constructs by ELISA experiment and semi-quantitative PCR detection.Conclusion Differential Proteomics found 52 different proteins that were at least 2 times stronger in supernatants of early-stage than ones of late-stage. Protein microarray technology found 6 growth factors secreted mainly at early stage by chondrocytes-scaffold construct. The chondrogenic induction effects of these proteins still need to be investigated.4 Effects of IGFBP4 and IGFBP6 on chondrogenic differentiation of BMSCs in vitroMethods P3 BMSCs were centrifuged into pellets and were divided into four groups: Control 1 group with medium containing transformation growth factor beta 1 (TGFβ1); Combined induction group with medium containing 100ng/ml IGFBP4, 100ng/ml IGFBP 6 and TGFβ1; IGFBP induction group with medium containing 100ng/ml IGFBP4 and 1 OOng/ml 6; Control 2 group with routine culture medium without TGFβ1 or IGFBP. At day 20, HE, toluidine blue and type II collagen immunohistochemical staining were tested and the expression of cartilage-specific genes were observed by semi-quantitative polymerase chain reaction (PCR) analysis.Results At day 20 cartilage-like tissues could be seen at the rim of pellets with cartilage lacunas in Control group 1 and Combined induction group, but not be seen in pellets in Control group 2 and IGFBP induction group by HE staining, toluidine blue staining and type II collagen (ColⅡ) immunohistochemical staining. Cartilage-specific genes, ColⅡand Aggrecan, were expressed in pellets of all the four groups. Sox9 was only expressed in Combined induction group and in Control group 1, but not in IGFBP induction group and Control group 2. Expression of Aggrecan and Sox9 was stronger in Combined induction group than that in Control group I, which implied an positive improving effects of IGFBP4,6 on chondrogenic differentiation of BMSCs.Conclusion 100ng/ml of IGFBP 4,6 cannot direct BMSCs into chondrocyte linage alone, but can promote chondrogensis by combination with TGFβ1.