Adhesion, Proliferation And Differentiation Of Mouse Mesenchymal Stem Cells On The Functionalized Self-assembling Peptide Hydrogel Scaffold In Vitro

Bone graft is a common method for repairing bone defects in clinic.Autograft bone is the gold standard for repairing large bone defects,but the application is limited for the shortage of sources,resulting in new bone defect and pain. The rapid development of bone tissue engineering has brought a new choice for the repair of bone defects.At present, scaffold, one of the three elements of bone tissue engineering, has become a hot research. The appearance of many kinds of scaffolds can provide more choice for repairing bone defects. However, there are also many problems. Allogeneic bone has a natural bone tissue structure, but may cause immune reaction and spread disease. Artificial synthetic material can be a lot of production, but there are many shortcomings, such as poor hydrophilicity, poor cell adhesion, insufficient osteoinductive properties.The self assembling peptide was artificial synthesized, which components are amino acids, can spontaneously assemble into nano fibers and form a network structure. Fiber diameter is 10 ~ 20 nm, the pore size is 5 ~ 200 nm,and the water content is more than 99%. The hydrogel can be rapidly formed by adding ions into the self assembling peptide solution. The advantages of this hydrogel scaffold are: 1, The hydrogel has a similar micro environment, which is suitable for cell adhesion, proliferation and differentiation. 2, The composition is clear, the degradation product is short peptide or amino acid,whth good biological compatibility. 3, They have a certain plasticity. RADA16- I is a classical self assembling peptide, the sequence is Ac N-RADARADARADARADA-CONH2,and there are many reports on the growth of cell on RADA16- I hydrogel. Recently, a number of scholars have used different functional short peptide to modify RADA16- I, and the research found that these new functional self assembling peptide hydrogel scaffolds have good effect on cell adhesion, proliferation and differentiation.The DGEA(Asp-Gly-Glu-Ala) sequence from collagen I was able to function on the α2β1 integrin receptor, which promotes the cell adhesion. We have synthesized the new functional self assembling peptide RAD-DGEA by combining RADA16- I C ternimal and DGEA by solid phase method,and The sequence is Ac N-RADARADARADARADA-GGDGEA-CONH2.When overving its microstructure, it is found that it can only form a short fiber and can not form a network structure. We cultured C57 mouse Bone marrow mesenchymal stem cells(MSCs) on the blank slide, hydrogel RADA16-Ⅰhydrogel and DGEAmx hydrogel, and detected cell adhesion, proliferation and differentiation, in order to get an ideal scaffold for bone repair.Objective:⑴ To observe the micro structure of self assembling peptide by atomic force microscopy(AFM).⑵ To detect the cell adhesion on the scaffolds by Cell counting method.⑶ To detect the cell proliferation on the scaffolds by CCK-8.⑷ To evaluate the cell osteogenic differentiation: by detecting alkaline phosphatase(ALP) activity, intercellular adhesion molecule Ⅰ(ICAM-Ⅰ),and osteocalcin(OCN).Methods:⑴ Observing the microstructure of self assembling peptide: the peptide solution was prepared and then added to the surface of the fresh mica plate, and the microstructure was observed under the atomic force microscope after natural drying.⑵ To detect the cell adhesion on the scaffolds by Cell counting method under microscope: place the sterile coverslips in 24 well plates and the self assembling peptide solution is dripped on the coverslips to prepare hydrogel. The MSCs suspension was added to the surface of hydrogel.The cells were photographed and randomly selected 3 fields of vision for cell counting.⑶ To detect the cell proliferation on the scaffolds by CCK-8: MSCs were cultured on the scaffold and proliferation was detected using CCK-8 kit.⑷ Cytoskeleton detection: observed and photographed cytoskeleton stained with phalloidin and nucleus stained with DAPI under laser scanning confocal microscope.⑸ To evaluate the cell osteogenic differentiation: cells were cultured on scaffold in osteogenic induction medium. The ALP activity was detected by using alkaline phosphatase activity kit.ICAM-Ⅰand OCN were detected by real-time PCR.Results:⑴ The DGEA peptide could not form fibres. RADA16-Ⅰcould form homogeneous long fibres and then formed network. RAD-DGEA could only form short fibers, but the DGEAmx,the Mixture of RAD-DGEA and RADA16- Ⅰ,could form fibre network as RADA16-Ⅰ. According to the result of AFM, The scaffolds for the following experiments were divided into 3 groups: blank slide, RADA16-Ⅰand DGEAmx.⑵ The number of MSCs that adhered to the scaffolds was managed by statical method. There was no significant difference between groups at 10 th minutes(P > 0.05).At 30,60,90 th minutes there were significant differences between groups(P<0.05).⑶. Proliferation of MSCs on three groups of scaffolds was managed by statical method. There was no significant difference between the three groups after 1 days of culture(P > 0.05). Proliferation of MSCs on the GEAmx scaffold was higher than that on the blank slide after 3 days of culture(P<0.05). There were significant differences between the 3groups after 5 days of culture(P < 0.05). Proliferation of MSCs on the DGEAmx scaffold was higher than that on the other groups after 7 days of culture(P < 0.05).Conclusion;Scaffold is one of the three essential elements of bone tissue engineering and there are many requirements on the performance of it. Self assembling peptide as a synthetic material, has the advantages of being capable of mass production.Its hydrogel has a structure similar to that of natural extracellular matrix(ECM), which has high water content and porosity, that is suitable for cell attachment and growth. Because of its synthetic material is amino acid, the degradation products are non-toxic, can avoid the inflammation and immune reaction. In this study, DGEA was added to RADA16-Ⅰ by solid phase synthesis method and DGEAmx hydrogel scaffold was structured. The microstructure of self assembling peptide was observed, and the adhesion, proliferation and differentiation of MSCs on hydrogel scaffolds were detected. The results show that the DGEAmx hydrogel is a new type of scaffold for cell culture, and it has a good application prospect in bone tissue engineering.