| The development of bone tissue engineering technology has provided a new way for the clinical treatment of bone defects,but the existing problems of traditional seed cells such as mesenchymal cells and osteoblasts,such as insufficient number,limited amplification ability and easy aging,need to be solved.Human induced pluripotent stem cells(hi PSCs)have the potential of infinite self-renewal and multidirectional differentiation,and avoid ethical problems.Therefore,hi PSCs are undoubtedly the most ideal seed cell source for bone tissue engineering.Many small molecular compounds or growth factors have been reported to improve the osteogenic differentiation efficiency of hi PSCs,but the differentiation efficiency remains to be improved.In addition to biochemical regulation,it is necessary to use biomaterials to simulate the extracellular matrix and promote cell proliferation and differentiation efficiency by improving the cell microenvironment.Roughness has been widely used to regulate the osteogenic differentiation of ordinary stem cells such as mesenchymal stem cells in vitro.However,the adhesion of hi PSCs requires special surface modifications that have a large impact on the perception and subsequent adhesion process of cells.Therefore,it is of great significance to develop a modification method to support the adhesion of hi PSCs with adjustable roughness and to study the influence of cultured surface roughness on hi PSCs behavior in vitro.Synthetic surfaces,especially polypeptide modified surfaces,are considered ideal for mass culturing hi PSCs.We developed a synthetic surface of polydopamine(PDA)and grafted VN polypeptide derived from vitronectin to maintain hi PSCs self-renewal for more than 20 passages.In addition,previous studies of our research group have found that heating and stirring can promote the polymerization of dopamine,and control the polymerization time of dopamine can obtain the polydopamine coating with roughness from nanometer to submicron.Therefore,on the basis of these work,In this study,polydopamine coating with different polymerization time was prepared on the surface of polystyrene.After modification by carboxymethyl chitosan and peptides,a rough polypeptide modified coating supporting the adhesion of hi PSCs was constructed to study the effect of surface roughness on in vitro adhesion,pluripotent maintenance and osteogenic induced differentiation of hi PSCs.Considering that compared with the widely used Matrigel,the cell adhesion and self-renewal performance on the existing peptide modified surface still needs to be improved,especially the research consensus that rough surface is not conducive to stem cell adhesion.In this study,a new peptide with better performance was developed by changing the amino acid sequence,laying a foundation for the subsequent research.First,two novel peptides with sequences of NP1 peptide(AcKGGTYRAYRGDVFTMP)and NP1 peptide(Ac-KGGVFTMPRGDTYRAY)were successfully designed from VN peptide(Ac-KGGPQVTRGDVFTMP)and BSP peptide(Ac-KGGNGEPRGDTYRAY)that were reported to have good adhesion properties by cleaving and recombining their near-D amino acid sequences.Experimental results confirmed that NP2 peptide had better supportive cell adhesion and long-term self-renewal function compared with VN peptide control group.Antibody blocking experiment confirmed that the main integrin receptor of NP2 peptide was ?V?3.Further quartz crystal microbalance quantitative measurements confirmed that NP2 peptide has stronger ?V?3 receptor affinity than VN peptide.These work laid a foundation for the subsequent preparation of rough peptide modified coatings and the study of cell behavior regulation.Further,using the method previously reported by our research group,carboxymethyl chitosan and NP2 polypeptide were grafted onto polystyrene surfaces in dopamine hydrochloride solution at 60°C at 300 rpm for different times(10 min,1h,12 h and 24 h).Atomic force microscopy(AFM)results confirmed the successful preparation of polydopamine coating with nanometer to submicron roughness and the final polypeptide modified culture substrate.Water contact angle measurement showed that each surface showed similar hydrophilicity,and FITC labeled peptide quantitative measurement confirmed that there was no significant difference in peptide graft amount.Further,hi PSCs of skin or urine origin(f-hi PSCs and u-hi PSCs)were inoculated on different roughness surfaces,the results of CCK8 and crystal violet showed that submicron rough surface(Ra= 180 nm)was not conducive to adherent and proliferation of f-hi PSCs.However,the more easily differentiated u-hi PSCs could not adapt to our surface culture.Meanwhile,RT-PCR results also showed that the pluripotent genes expression of f-hi PSCs was up-regulated at nanorough surface(Ra= 17 nm),indicating that relatively smooth surface was conducive to self-renewal of f-hi PSCs.Further,the adhesion related genes were examined and the results confirmed that rough surface may affect the adhesion and multipotency of hi PSCs by regulating FAK,Vinculin,E-adherin and MYH9 genes.In addition,we investigated the effect of roughness on osteogenic differentiation of hi PSCs by nanorough and micro-rough surfaces(Ra= 6.4 nm and 180 nm,respectively).Alizarin red staining showed that nanorough surface(Ra= 6.4 nm)was more conducive to calcium deposition during osteogenesis.Flow cytometry,RTPCR and immunofluorescence results also showed that nanorough surface(Ra= 6.4 nm)was more conducive to osteogenic differentiation of hi PSCs.In this research,the effect of amino acid sequence on the performance of polypeptide was studied and the novel polypeptide sequence obtained has a good application prospect.More importantly,the effects of polypeptide modified surface roughness on hi PSCs growth and osteogenic differentiation in vitro were studied,and the related molecular mechanisms will be further studied.These findings are of great significance for guiding the design of biomaterials and improving the efficiency of osteogenic differentiation of hi PSCs. |
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