Preparation Of Biomimetic Hybrid Hydrogel And Its Application In 3D Culture Of Bone Marrow Mesenchymal Stem Cells In Vitro

Extracellular matrix(ECM)provides structural support totissue cells,protects and nourishes them,andhas an important impact on cell proliferation,differentiation and other life activities.Hydrogel which has high water content,excellent transport properties of nutrients and similarmechanical properties as the natural ECM,is an ideal cell 3D culture platform.Although the synthetic polymer hydrogels have good biocompatibility,controllable structure and property,they are poor in biological activity,and need to be modified by biologically active molecules to improve their degradation performance,promote cell growth and differentiation.In this study,rabbit bone marrow mesenchymal stem cells(RBMSCs)were isolated,and thermo-sensitive hydrogel system of Pluronic F127 was prepared.In order to mimic the natural extracellular matrix,we synthesised collagen mimetic peptide(CMP),matrix metalloproteinase(MMP)degradable peptide and constructed bionic hybrid hydrogels with multi-arm PEG.The hydrogel was used for the 3D culture of RBMSC,and the cytocompatibility of hydrogels and the effect on cell differentiation were evaluated,to provide the basis to develop new bioactive hydrogels system.The main contents and results of this work were as follows:1.Pluronic F127 hydrogel:preparation and 3D culture of RBMSCsThe RBMSCs were isolated and identified as having multidirectional differentiation potential.The thermosensitive hydrogel of Pluronic F127 was prepared and the suitable gelation conditions were determined.The hydrogel was porous and the mechanical strength was high.With increased concentration of the hydrogel,the mechanical strength enhanced,and the solution-gel phase transition temperature reduced.The hydrogel system was used to embed RBMSCs for 3D culture with good cell compatibility,but no significant effect on cell differentiation,which wasattributed to the poor bioactivity of the hydrogel system.2.Collagen mimetic peptide-PEG hybrid hydrogel for 3D culture of RBMSCsThe collagen mimetic peptide(GPO)8-CG-RGDS was designed and synthesized.The circular dichroism spectra of the CMP showed that the CMP could spontaneously assemble into triple-helix conformation similar to natural collagen,and the thermal denaturation temperature was about 49.4 ?.A bionic CMP-PEG hybrid hydrogel which had moderate mechanical strength and porous internal structure was fabricated by the conjugation of CMP and 4-arm PEG-MAL.After 24 hours of culture in the hybrid hydrogels,most of the RBMSCs remained viable,indicating that the hybrid hydrogels had good cell compatibility.The detection of gene expression suggested that the 3D environment of hybrid hydrogel was benefical to cartilage differentiation of RBMSCs.3.MMP-degradable peptide-PEG hybrid hydrogel for 3D culture of RBMSCsMMP-degradable peptide with cysteine at both ends was synthesized,and conjugatedwith 4-arm PEG-MAL to fabricate a bionic MMP-degradable peptide-PEG hybrid hydrogel.The gelation reaction was rapid,and the concentration of the gelatinous precursor solution affected the internal pore size and mechanical strength of the gel.The hybrid hydrogel was applied to 3D culture of RBMSCs.Preliminary studies had shown its good cell compatibility,and provided a basis for further study of cell-mediated degradation of the hydrogel system and its effects on cell differentiation.