Study On Three-dimensional Culture Of Human Amniotic Mesenchymal Stem Cells In Peptide Hydrogel

Objective To investigate the growth,proliferation,expression of stemness-related genes and osteogenic differentiation of human amniotic mesenchymal stem cells in peptide hydrogels.Methods1)The hydrogel RGD(Ac N-RADARADARADARADA-GG-RGDSP-CONH2),TTS(Ac N-RADARADARADARADA-GG-TTSWSQ-CONH2),FOG(Ac N-RADARA DARADARADA-GG-FOGER-CONH2)were examined by transmission electron microscopy,circular dichroism spectroscopy and rheological analysis;1% of the above three peptides were sequentially mixed with 1% RADA16 at the ratio of 3: 7,3: 7 and 2: 8 respectively,sonicated and stored at 4?.2)hAMSCs were isolated by two-step digestion,cultured and purified to third passage.h AMSCs were identified by flow cytometry,immunocytochemical staining,cell morphology and directional differentiation.h AMSCs were appllied to subsequent coculture experiments with self-assembling peptide hydrogels at third passage.3)The experiment was divided into five groups:2D group(2D Group,2D),G1 group(pure RADA16),G2 group(RAD/RGD,RADA16: RGD=3:7),G3 group(RAD/TTS,RADA16: TTS =3:7)and G4 group(RAD/FOG,RADA16: FOG=2:8);Establishment of h AMSCs and the G1,G2,G3 and G4 groups of hydrogel scaffold complexes as well as two-dimensional control model;Inverted phase contrast microscope was used to observe the cell morphology of each group;Survival cells in scaffolds was examined using Live/Dead;CCK-8 and Ed U were used to detect the proliferative activity of the cells in the scaffold respectively;Flow cytometry was used to detect the cell cycle in each group;Subsequently,alizarin red staining was used to evaluate the abilitiy of the osteogenic differentiation;The m RNA expressions of stemness-related genes in h AMSCs,including Nanog,Oct-4a and Rex-1,were analyzed by q PCR at day 8 after hydrogels and 2D culture,and the levels of osteoblast differentiation-related genes,including Runx2,Osx,BSP,ALP,Ocn and Col1?1,were investigated at day 7,14 and 21.Results1)the RADA16-designed peptide mixtures could self-assemble into a homogeneous,transparent and elastic hydrogel quickly.Transmission electron microscopy(TEM)confirmed that self-assembling peptide scaffolds were in the form of nanofiber dimension in aqueous solution;Circular dichroism spectra and Transmission electron microscopy analysis revealed the three designed peptides formed the second structure of typical ?-sheet in aqueous solutions and spontaneously assembled into well-defined nanofibers with width close to 7 nm and lengths ranging from hundreds of nanometers to several micrometers;Rheological examination revealed that the storage modulus(G')of the hydrogel was higher than the loss modulus(G"),indicating that these peptides behaved as elastic peptide scaffolds rather than solutions.2)The h AMSCs displayed typical mesenchymal morphology with long cord-like and spiral-shaped growth at 3th passage.CD44,CD73,CD90,CD105 and vimentin were upregulated in the h AMSCs,but CD34,CD19,CD45,CD11 b and HLA-DR were downregulated;Alizarin red staining showed mineralized nodule formation in the osteoblast induced cells;Lipid droplets could be visualized in the cytoplasm by Oil Red O staining after adipogenic induction;Toluidine blue staining revealed that a large amount of glycosaminoglycans were secreted into the cell matrix upon chondrogenic induction.3)The Live/Dead assay reveled that more than 80% of h AMSCs encapsulated within the hydrogel survived in each group;CCK-8 and Ed U experiments showed that the proliferation of h AMSCs in hydrogels were slower than that on 2D Petridish,and this might be attributed to the resting state of h AMSCs in hydrogel.Cell cycle analysis demonstrated that more than 70% of the h AMSCs in the hydrogels were tended to be in the G0/G1 phase,and about 21% of the h AMSC were to be in the S phase.Ed U data showed that the proliferative rate of G1> G2> G3> G4 was consistent with the cell cycle analysis.Finally,compared with 2D group,the hydrogels could upregulate m RNA expression of stemness-related genes(Nanog,Oct-4a and Rex-1)and osteogenic differentiation genes(Runx2,Osx,BSP,ALP,Ocn and Col1?1).Conclusion1)The proliferation of h AMSCs encapsulated within the peptide hydrogels were slower than that on the traditional 2D Petridish.2)After 8 days of culture,the m RNA levels of the stemness-related genes were upregulated in the h AMSCs encapsulated within the hydrogels.3)After osteoinduction,the m RNA levels of h AMSCs encapsulated within the peptide hydrogels were also up-regulated.