摘要
BACKGROUND:Growth differentiation factor 5 is a member of the transforming growth factor superfamily and one of the earliest markers of joint development.Growth differentiation factor 5 has an important role in cartilage repair. OBJECTIVE:To explore the action mechanism of growth differentiation factor 5-induced chondrogenic differentiation of bone marrow mesenchymal stem cells. METHODS:Rabbit bone marrow mesenchymal stem cells were isolated and cultured.CCK-8 assay was used to detect the effect of different mass concentrations of growth differentiation factor 5 on the proliferation activity of bone marrow mesenchymal stem cells.RT-PCR was utilized to detect the expression of genes related to chondrogenic differentiation of bone marrow mesenchymal stem cells induced by different mass concentrations of growth differentiation factor 5.To further investigate the action mechanism of growth differentiation factor 5-induced chondrogenic differentiation of bone marrow mesenchymal stem cells,we added inhibitor XAV-939 and activator Laduviglusib of Wnt/β-catenin signaling pathways to induce cell culture for 14 days.RT-PCR and western blot assay were performed to detect the expression of cartilage-related genes and Wnt/β-catenin signaling pathway proteins. RESULTS AND CONCLUSION:(1)CCK-8 results showed no significant effect of growth differentiation factor 5 on the proliferation of bone marrow mesenchymal stem cells.(2)Growth differentiation factor 5 promoted the expression of cartilage-related genes type Ⅱ collagen,aggrecan and Sox9,among which growth differentiation factor 5 induced a significant upregulation of cartilage-related genes in the 50 ng/mL group.(3)Addition of Laduviglusib,an activator of Wnt/β-catenin signaling pathway,upregulated Sox9,β-catenin and type Ⅱ collagen expression(P<0.05).Addition of XAV939,an inhibitor of Wnt/β-catenin signaling pathway,down-regulated Sox9,β-catenin and type Ⅱ collagen expression(P<0.05).(4)Taken together,growth differentiation factor 5-induced chondrogenic differentiation of bone marrow mesenchymal stem cells may be associated with the activation of the Wnt/β-catenin signaling pathway.
摘要
BACKGROUND:Natural bone morphogenetic protein 2 disperses and degrades rapidly in vivo,reducing local concentration and therapeutic efficacy.Simply combining bone morphogenetic protein 2 with tissue engineering scaffolds could not stay in vivo for a long time,making it difficult to achieve good sustained and controlled release effects.OBJECTIVE:To prepare and test the biological properties and chondrogenic induction effect of collagen-binding domain-bone morphogenetic protein 2-collagen cartilage scaffold.METHODS:SD rat tail collagen was extracted and a collagen cartilage scaffold was prepared using a vacuum freeze-drying machine chemical crosslinking method.The plasmid expressing collagen-binding domain-bone morphogenetic protein 2 was constructed by rapid cloning C112 homologous recombination,constructed by genetic engineering,and introduced into E.coli,and then collagen-binding domain-bone morphogenetic protein 2 was isolated and purified.Natural bone morphogenetic protein 2 and collagen-binding domain-bone morphogenetic protein 2 were combined with collagen cartilage scaffolds,respectively,to detect the release level of bone morphogenetic protein 2 in the scaffolds.The biocompatibility of collagen-binding domain-bone morphogenetic protein 2-collagen cartilage scaffold was detected by CCK-8 assay and F-Actin staining.Bone marrow mesenchymal stem cells were implanted on two kinds of collagen cartilage scaffolds for chondrogenic induction,and their chondrogenic induction activity was tested.RESULTS AND CONCLUSION:(1)The binding rate of collagen-binding domain-bone morphogenetic protein 2 to collagen cartilage scaffolds was higher than that of natural bone morphogenetic protein 2(P<0.05).After being immersed in PBS for 7 days in vitro,the release of bone morphogenetic protein 2 in the collagen-binding domain bone morphogenetic protein 2-collagen cartilage scaffold was smaller than that in the natural bone morphogenetic protein 2-collagen cartilage scaffold(P<0.05).The results of the CCK-8 assay and F-Actin staining showed that the collagen-binding domain-bone morphogenetic protein 2-collagen cartilage scaffold had no obvious cytotoxicity and had good biocompatibility.(2)After 14 days of chondrogenic induction,ELISA detection demonstrated that the expressions of agglutincan and type Ⅱ collagen A1 in the collagen-binding domain-bone morphogenetic protein 2-collagen cartilage scaffold group were higher than those in the natural bone morphogenetic protein 2-collagen cartilage scaffold group(P<0.05).Under scanning electron microscopy,more bone marrow mesenchymal stem cells were observed on the inner wall of the pores of the two groups of scaffolds,and the cell morphology and size were the same,and the cells were closely arranged,without cell fragmentation or abnormal morphology.(3)The results indicate that the collagen-binding domain-bone morphogenetic protein 2-collagen cartilage scaffold has good biological properties and chondrogenic induction activity.