An Experimental Study Of Regulation Of VEGF And Notch Signaling Pathway On Osteoarthritis Cartilage Repair By Placenta-derived Mesenchymal Stem Cells

BackgroundOsteoarthritis is a common orthopaedic disease and a higher incidence in the elders. The major pathological change of oateoarthritis is degeneration of articular cartilage. The cartilage indicates a poor ability to repair by itself because there is no blood supply, nerve and lymphoid tissue distributed in it. The main symptoms of osteoarthritis is joint pain and activity limitation. It affects the daily life of patients, or even lead to loss of ability to work and induces families and serious social economic burden. But therapies for osteoarthritis are limited. The most effective method is joint replacement, but it requires expensive operation cost and prosthesis loosening and other complications are inevitable. Discovering a simple and effective method for osteoarthritis would be the benefits in social and economic sense.Mesenchymal stem cells(MSCs) derived from mesodermal stem cells, are pluripotency, widely used in the tissue engineering and regenerative medicine. It shows a promising future in cartilage repair by tissue engineering technology. Some researches have shown that intra-articular injection of MSCs can repair osteoarthritis cartilage defects. MSCs differentiate to chondrocytes is the key to treat osteoarthritis cartilage defect. It will promote repair effect of articular cartilage defect if promote the chondrogenic differentiation capacity of MSCs. Placenta-derived mesenchymal stem cells(PMSCs) is a kind of MSCs that derived from placenta tissue. Because derived from embryonic tissue, it has a higher proliferation activity and differentiation capacity. There will be a better result if PMSCs are applied for osteoarthritis cartilage repairing.Studies have shown that during the development of embryonic limb, inhibition of vascular endothelial growth factor(VEGF) expression can promote embryonic stem cell differentiate into cartilage tissue, and in adult tissue derived stem cells research has also shown that inhibition VEGF signaling expression can promote chondrogenic differentiation, and promote the repair effect of osteoarthritis cartilage defect. There are several signaling pathways involved in the process of chondrogenic differentiation of stem cells, the most important is the Notch signaling pathway, but the mechanism is not clear. Thus we envision the initiative to regulate Notch signaling pathway, observe the influences on chondrogenic differentiation of PMSCs, and find out the regulatory method which can promote PMSCs chondrogenic differentiation. And combine regulation VEGF signaling pathway with Notch signaling pathway approach to promote PMSCs chondrogenic differentiation, apply destabilization of the medial meniscus(DMM)osteoarthritis animal models in further step and observe its influences on cartilage repairing. ObjectiveThe main purpose of this study was to observe the effect of different Notch signaling pathway regulating method on PMSCs chondrigenic differentiation and find out the method which can promote differentiation capacity. And combined regulate VEGF and Notch signaling pathway, observe the chondrogenic differentiation capacity of PMSCs. Building a mouse model of knee osteoarthritis, apply the intra-articular injection of PMSCs which were regualated with VEGF and Notch signaling pathways, and observe the effect to articular cartilage defect repair, provide new evidence for the treatment of osteoarthritis. This study is divided into three parts:Part I: The effect of regulation of VEGF and Notch signaling pathway on the chondrogenic differentiation of PMSCs Methods(1) Culture PMSCs and analyse the surface markers of CD29, CD34, CD73, CD90, CD105 and CD166 by flow cytometry.(2) To observe the multipotency of PMSCs, osteogenic differentiation, adipogenic differentiation and chondrogenc differentiation was induced respectively. Alkaline phosphatase staining, Aizarin red staining and the gene expression of Runx2 and Osteocalcin were used to identify the osteogenic differentiation. Oil red O staining, PPARgamma and C/EBPa gene expression levels were used to identify the adipogenic differentiation. Alcian blue staining, type II collagen(Col-II) immunohistochemical staining and Aggrecan, Col-II, Sox9 gene expression levels were used to identified the chondrogenic differentiation.(3) Apply VEGF receptor inhibitor SU5416 as VEGF signaling pathway inhibitor, observe the chondrogenic differentiation of PMSCs.(4) Use Notch signaling pathway inhibitor LY411575 to inhibite Notch signaling pathway and Notch signaling pathways protein ligand Jagged1 to activate Notch signaling pathway, observe its effect on chondrogenic differentiation of PMSCs. According to the different treatment, the experiment is divided into six groups:(1)Control group: the normal chongdrogenic differentiation medium;(2)Notch 7d group: add LY4115757 to the medium in the initial 7 days of chondrogenic differentiation process;(3)Notch 21 d group: add LY4115757 to the medium in the whole process of chondrogenic differentiation(21 days);(4)Jag1 7d group: add Jagged1 protein to the medium in the initial 7 days of chondrogenic differentiation process;(5)Jag1 21 d group: add Jagged1 protein in the whole process of chondrogenic differentiation;(6)J7+N14 group: add Jagged1 protein in the initial 7 days and add LY411575 in the last 14 days.(5) Regulate VEGF signaling pathway together with Notch signaling pathway, observe the effect on chondrogenic differentiation of PMSCs. Experiments are divided into 4 groups:(1)Control group;(2)VEGFRi group : add SU5416 to the chondrogenic medium;(3)Jag1 7d group: add Jagged1 protein to the medium in the initial 7 days;(4) VEGFRi+Jag1 7d group : add SU5416 to the medium and add Jagged1 protein to the msdium in the initial 7 days. Results(1) Flow cytometry confirmed that PMSCs were positive for CD29, CD73, CD90, CD105 and CD166, positive rate were 100%, 100%, 100%, 54.7%, 93.1% respectively, and negative for CD34, positive rate was 0%.(2) PMSCs can successfully differentiate into osteoblast, adipocyte and chondrocyte, confirmed the multipotency of PMSCs.(3) Alcian blue staining positive zones and Col-II protein expression levels of VEGFRi group were higher than the Control group. Col-II, Aggrecan and Sox9 gene expression level were all significant higher than the Control group. Confirmed that inhibit VEGF signaling pathway promote the chondrogenic differentiation of PMSCs.(4) Notch 7d group, Notch 21 d group and Jag1 7d+Notch 14 d group significantly inhibited PMSCs chondrogenic differentiation. There were no no significant difference between Jag1 21 d group and Control group. The chondrogenic differentiation capacity of Jag1 7d group was significantly higher than the Control group. Confirmed that activate Notch signaling pathway in the initial stage can promote chondrogenic differentiation of PMSCs, and inhibite Notch signaling pathway can inhibit chondrogenesis.(5) The chondrogenic differentiation level in VEGFRi+Jag1 7d group was significantly higher than the VEGFRi group and the Jag1 7d group(P<0.05). Confirmed that combined regulation of VEGF signaling pathway and Notch signaling pathway plays a synergstic effect on chondrogenic differentiation of PMSCs.Part II: Construction of mouse knee joint osteoarthritis model Methods(1) Destabilization of the medial meniscus(DMM): The joint capsule immediately medial to the patellar tendon was incised with a blade and the joint capsule opened with micro-scissor. Isolate surrounding soft tissue, cut the medial meniscotibial ligament, then cut the entire medial meniscus.(2) Specimen processing: Mice were euthanized at 4 weeks, 8 weeks and 12 weeks post-operatively. Bilateral lower limb were fixed in formalin for 72 hours. Whole knee joint were decalcified for 2 weeks. Joints were embedded in paraffin and 5 mm frontal sections were taken.(3) Identification of results: H&E staining for analysing the knee structure, Alcian blue staining and Safranin O staining for analysing the cartilage matrix; OARSI histological score for assessing the degree of osteoarthritis; Col-II and Aggrecan immunohistochemical staining for evaluating cartilage matrix protein expression; anti-tartaric acid phosphatase(TRAP) staining for assessing osteoclasts activity in the subchondro bone; osteophyte size score and osteophyte maturity score for analysing osteophyte formation. Results(1) Histology analysis of knee joint: After 4 weeks of DMM, the articular cartilage has been worn and proteoglycan in the cartilage was reduced, but the articular surface is complete, showing early changes of osteoarthritis. After 8 weeks of DMM, the articular cartilage surface is incomplete, there is less cartilage remnants and less proteoglycan content in articular cartilage, showing typical changes of osteoarthritis. After 12 weeks of DMM, there was almost no cartilage left in tibial platform, and sclerosis of tibial platform subchondral bone showed severe osteoarthritis changes.(2) OARSI score of DMM 4 weeks model was 3±0.6, that was significant higher than the normal group(P<0.05); OARSI score of DMM 8 weeks model was 10.8±1.3, significant higher than the 4 weeks group(P<0.05); OARSI score of DMM 12 weeks model group was 15.8±1.2, significant higher than the 8 weeks group(P<0.05); Confirmed that DMM model is progressive degeneration.(3) In the normal articular cartilage, the protein expression levels of Col-II and Aggrecan were high, with the development of DMM model, the protein expression level of Col-II and Aggrecan were decreased progressively in cartilage matrix.(4) The number of osteoclasts in the subchondro bone got peak at 4 weeks after DMM then began to decline, and decreased to the baseline at 12 weeks after DMM.(5) There was osteophyte formation around tibia plateau after 4 weeks of DMM operation, but the osteophyte was small and the maturity was low. With the development of osteoarthritis model, osteophyte get large and mature.Part III: The animal experiment of regulation of VEGF and Notch signaling pathway on osteoarthritis cartilage defect repair by PMSCs Methods(1) DMM model: Same to part II.(2) Intra-articular injection of PMSCs: Intra-articular inject PMSCs after 4 weeks of DMM procedure once a week. Depending on the different treatment of PMSCs, experiment was divided into 5 groups:(1)Control group: intra-articular inject medium;(2)PMSCs group: intra-articular injection PMSCs;(3)P+V group: intra-articular inject PMSCs+SU5416;(4)P+J group: intra-articular inject PMSCs+Jagged1;(5)P+V+J group: intra-articular inject PMSCs+SU5416+Jagged1.(3) Track PMSCs use CM-Dil: PMSCs were labled use CM-Dil reagent, observe the survival ability of intra-articular injected PMSCs.(4) Specimen processing: Harvest the specimen at 4 and 8 weeks after intra-articular injection treatment respectively. Then the specimen were fixed, decalcified and sliced.(5) Identification of results: H&E staining for analysing the knee structure, Alcian blue staining and Safranin O staining for evaluating the repaired cartilage matrix; OARSI histological score for assessing the degree of osteoarthritis; Col-II, Aggrecan and Col-X immunohistochemical staining for analysing the protein expression in repaired cartilage matrix; TUNEL staining for analysing apoptosis of articular chondrocyte. Results(1) 4 weeks after intra-articular injection, the CM-Dil labeled PMSCs were still visible in the articular cavity, confirmed that PMSCs injected into articular cavity can survive for a long time.(2) Histology analysis of knee joint: After 4 weeks and 8 weeks treatment, the repaired cartilage can be found in the PMSCs group, but the repaired cartilage layer was thin, and the cartilage matrix was rare, but the articular surface was smooth, no cartilage crack; the repaired cartilage in P+V group and P+J group were better than PMSCs group, the cartilage layer were thicker and there were more cartilage matrix; the cartilage repair effect of P+V+J group is best, hyaline cartilage was thick, there was more cartilage matrix in the repaired cartilage. OARSI score also confirmed the same results.(3) The protein expression level of Col-II and Aggrecan in the cartilage of PMSCs group was low, but the expression level of cartilage degeneration marker Col-X was high, confirmed the poor quality of repaired cartilage. Col-II and Aggrecan protein expression level in P+V group and P+J group were higher than PMSCs group, and the Col-X protein expression level were lower, confirmed the repaired cartilage of P+V group and P+J group were better than PMSCs group. However, the expression level of Col-II and Aggrecan in P+V+J group were higher than the other groups, and Col-X expression level was lower than other groups, confirmed the best quality of repaired cartilage in P+V+J group, which was close to the normal cartilage.(4) The amount of TUNEL staining positive chondrocytes in PMSCs group was less than the Control group, there was a statistically significant difference(P<0.05), confirmed intra-articular injection PMSCs can inhibit chondrocyte apoptosis. The number of TUNEL staining positive cells of P+V group and P+J group were significant less than PMSCs group(P<0.05). The number of TUNEL staining positive chondrocytes in P+V+J group was significantly less than other groups(P<0.05). Conclusions(1) PMSCs have multipotency; inhibit VEGF signaling pathway can promote the chondrogenic differentiation of PMSCs; activate Notch signaling pathway at the initial stage can also promote the chondrogenic differentiation; combined regulation of VEGF and Notch signaling pathway plays a synergistic effects on chondrogenic differentiation of PMSCs.(2) The DMM operation can successfully construct a mouse model of knee joint osteoarthritis, and the model was progressive development, it’s suitable for osteoarthritis research.(3) Intra-articular injection of PMSCs can repair the cartilage defect of osteoarthritis in mice, combined regulation of VEGF and Notch signaling pathway promotes the cartilage repair effect of PMSCs.