The Osteogenetic Efficacy Of Self-assembling Peptide RADA16/TRSAW Modified HA/?-TCP Based On SCR Technology

BackgroundAt present,in the case of bone defects caused by physical disruption and pathological resection,a large amount of bone grafting material is needed.Due to the limitation of autologous bone mass and the potential pathogenicity and immunogenicity of allogeneic bone,the repair of large bone defects is severe.Although the traditional tissue engineering strategy has achieved certain effects,the additional amplification steps of stem cells may bring about the risk of infection,ethical problems,application immediacy and high cost.In recent years,autologous bone marrow,which can be used with low immune rejection,has attracted the attention of clinicians.It can be applied to the repair of sudden bone defects,avoiding the cumbersome process and risk of in vitro expansion.Applying bone marrow directly inject to bone defects.stem cells are easily lost,resulting in unstable effects.Therefore,selective cell retention technology emerged.The core is to use the suitable mesh structure and surface adhesion function of the scaffold material to achieve enrichment of osteoblast-related stem cells and factors in the bone marrow rapidly.In summary,we propose to combine the selfassembled extracellular matrix material RADA16-? sequence with the osteogenic parathyroid hormone function peptide TRSAW sequence to construct the self assembling polypeptide RADA16/TRSAW,and modify the HA/?-TCP materials.The bioceramic scaffold effectively enhances the enrichment efficiency of selective cell retention technology by physically blocking and attracting cells and factors.Purpose1.Constructing an osteogenic active polypeptide,analyzing the physical properties and biological properties of the polypeptide,and initially evaluating the rationality as a modifying material.22.Constructing a polypeptide-modified HA/?-TCP functional scaffold material,and evaluate the biological performance of the scaffold by MSCs suspension liquid simulating stem cell retention technology,and lay a foundation for bone marrow SCR technology.3.To evaluate the enrichment efficiency and osteogenic effect of functional scaffolds under SCR technique by constructing a mouse femoral defect model.Method1.Analyze the microstructure of the peptide by atomic force microscopy,verify the secondary structure by circular dichroism,measure the charge of the material by ZATA potentiometer,verify the hydrophilic properties of the material by contact angle,and screen out the appropriate gel construction strategy for in vitro cells.2.Inoculate MSCs into the microenvironment formed by polypeptide gel,and evaluate the growth and adhesion of gel peptides to MSCs by morphological observation,real-time quantitative polymerase chain reaction(RT-PCR)quantification and Western blotting.Effects of osteogenic differentiation and angiogenesis.3.Gel-modified HA/?-TCP was used to form composite materials.The morphology of the modified materials was observed by environmental scanning electron microscopy(SEM).The biopsy of morphological observation,real-time quantitative polymerase chain reaction(RT-PCR)and Western blotting were used to evaluate the growth and adhesion of gel peptides to MSCs.The effects of attachment and osteogenic differentiation,as well as the mechanism of osteogenesis.Flow cytometry was used to analyze the enrichment effect of functional scaffolds on bone marrow cells by SCR technique.The skeletal model of bone defect in the middle femur of nude mice was constructed.To evaluate the repair effect of functional stents on bone defects under SCR technology.MicroCT were performed after 16 weeks.Result1.In the trigger of PBS solution,RADA16-?,TRSAWmax can form a gel form,while RADA16/TRSAW is a semi-gel state,comprehensively compare the three polypeptides into a fiber-like network structure,secondary structure,stability and hydrophilicity.RADA16-?,TRSAWmax group have a relatively stable ? secondary structure,fiber three-dimensional network structure,good hydrophilic properties,RADA16/TRSAW peptide physical properties are weaker than the first two groups2.CCK-8 reagent was used to detect the proliferation of cells in different materials at different time points.The RADA16-? and TRSAWmax groups were superior to the plate culture group,and the osteogenic induction medium was induced.after 3,7 and 14 days,qPCR,Western Blot and the alkaline phosphatase activity test kit was tested and osteogenic induction.The expression of Ang1 in the TRSAWmax functional scaffold material group was statistically different from that in the RDA16-? group.The supernatant of each group of MSCs was cultured for 3 days.The co-cultured EPC was taken out.The results showed that the vascular reticulum formation was obvious in the TRSAWmax group.The osteogenic induction of the TRSAWmax functional scaffold material group had higher gene expression levels and protein expression levels of Runx2,ALP,OPN and OCN.3.Under the trigger of PBS solution,RADA16-? and TRSAWmax hydrogels can be uniformly attached to the surface of TCP material by environmental scanning electron microscopy observed.After MSCs cell suspension was used to simulate selective cell retention,CCK-8 confirmed that TRSAWmax functional scaffolds retained more MSCs.The number of proliferating cells was statistically different at 5,7,and 9 days.After culture with osteogenic induction medium,gene expression levels and protein expression were evaluation by qPCR,Western Blot and alkaline phosphatase activity assay kits.The 7 and 14 days TRSAWmax functional scaffold material group had higher gene expression levels and protein expression levels of Runx2,ALP,OPN and OCN.Related osteogenic pathways can promote osteogenesis by Src regulation of Erk.4.By flow cytometry,the enrichment efficiency of TRSAWmax on nucleated cells,,CD90+/CD105+ double positive cells and CD34+ /CD31+cells in bone marrow was significantly higher than that of the simple material group.The model of the critical bone defect of the femur in nude mice was constructed.The repair effect of the implant material was evaluated by imaging.The results showed that the TRSAWmax group had better osteogenic ability in vivo.Conclusion1.Self-assembled peptide hydrogel is a good modification material.The experimental design adds functional short peptides on the basis of RADA16-?.On the one hand,it does not affect the RADA16-? structure by reasonable ratio.On the one hand,the biological properties of the hydrogel are increased,and the microenvironment is superior to the simple plate culture group for the proliferation,adhesion,osteogenic differentiation and angiogenesis of MSCs,which is an ideal modification material.2.The peptide hydrogel can be uniformly modified on the surface of TCP material to form a functional scaffold complex with TCP.The microenvironment constructed can induce MSCs to differentiate into osteoblasts,and its adhesion and proliferation trend.Significantly better than the simple material group.3.The functional scaffolds rapidly constructed by SCR technology focus on the enrichment of stem cells.The functional scaffolds rapidly constructed by SCR technology have significant effects on the repair of critical bone defects in nude mice.