Experimental Study On The Osteogenesis Effects Of A New-type Functional Self-assembling Polypeptide Hydrogel RGDmx

Part? Preparation of A New-Type Self-Assembled Polypeptide Hydrogel and Its PropertiesObjective:To prepare a new-type self-assembled polypeptide hydrogel and its properties.Methods:RADA16-I and RADA16-RGD polypeptide lyophilized powder were prepared into polypeptide hydrogel mother liquor,which were then prepared as a novel self-assembled peptide water Gel RGDmx by mixing RADA16-?and RADA16-RGD in a volume ratio of 4:1.The properties of the polypeptide hydrogels were evaluated by circular dichroism spectra,rheological property measurement,transmission electron microscopy and gelation observation.Results:A new-type self-assembled polypeptide hydrogel RGDmx was successfully prepared.The circular dichroism spectroscopy analysis showed that the polypeptide RADA16-RGD had a stable?-fold secondary structure.The rheological experiment showed that the increase of hydrogel concentration can improve the stability of hydrogel,and that the stability of hydrogel structure decreased with the increase of ionic strength.Transmission electron microscopy showed that the polypeptide RADA16-RGD could only form short fiber particles with uneven thickness,but RGDmx could form a long nanofiber structure similar to that of RADA16-?and finally merge as a three-dimensional network structure.The RGDmx fiber had larger diameter than RADA16-?,similar length to RADA16-?,and larger pore than RADA16-?.The RADA16-I and RGDmx were self-assembled into translucent gel-like substances,and RADA16-RGD could not self-assemble into gel.Conclusion:The novel self-assembled polypeptide RGDmx and the classical polypeptide RADA16-?can self-assemble to form a stable?-fold structure under the triggering of cations,can maintain their own morphology and also have certain mechanical strength.RGDmx can form a three-dimensional network of long nanofibers similar to RADA16-I.Part? A Novel Self-Assembled Polypeptide Hydrogel,RGDmx,Promotes the Osteogenic Differentiation of MC3T3-E1 CellsObjective:To observe the effect of the self-assembled polypeptide hydrogel RGDmx on the adhesion,proliferation and osteogenic differentiation of MC3T3-E1 preosteoblasts in mice.Methods:MC3T3-E1 cells were inoculated into the self-assembled polypeptide hydrogels RGDmx and RADA16-I for culture induction,and osteoblast-induced cells were cultured as controls.The adhesion growth of MC3T3-E1 cells on the surface of the hydrogel was observed by laser confocal microscopy 3 and 7 days after culture was induced.The cell proliferation activity was measured via the CCK-8 method at 1,3,5 and 7days after culture induction.The alkaline phosphatase activity was measured at 1,3,5,7,10 and 14 days after induction.Extracellular calcium deposition was observed by Alizarin Red S staining at 1 and 3 weeks after culture induction,and the expression of OPN?1w?and COL-I?3w?was detected by Western blotting.Results:Laser confocal microscopy showed that MC3T3-E1 cells grew well on the RGDmx polypeptide hydrogel and that the adhesion was higher than that of the RADA16-I and blank control groups.The CCK-8detection assay indicated that cell proliferation in the RGDmx group was higher than that in the RADA16-I group.The activity of alkaline phosphatase in the RGDmx group was higher than that in the RADA16-I and blank control groups.The expression of OPN?1w?and COL-I?3w?in the MC3T3-E1 cells in the RGDmx group was higher than that in the RADA16-I and blank control groups.Conclusion:The new-type self-assembled polypeptide hydrogel RGDmx can promote the adhesion,proliferation and osteogenic differentiation of MC3T3-E1 cells compared with the traditional RADA16-I hydrogel.PART ? Effect of A New-Type Self-Assembled Polypeptide Hydrogel RGDmx on Repair of Femoral Condylar Defects in SD RatsObjective:To observe the effect of RGDmx,a new-type self-assembled polypeptide hydrogel,on repair of femoral condylar defects in SD rats.Methods:SD rats were randomly divided into three groups?6 rats in each group?and animal models were established which had 2.7 mm bone defect on bilateral femoral condyles.PBS buffer,RADA16-I hydrogel and RGDmx hydrogel were implanted into femoral condylar defect areas of SD rat,and their specimen were then analyzed at 12w after operation.The basic condition of bone defect was determined by gross observation of the specimens,the new bone was detected by Micro-CT,and the osteogenesis effect was observed by HE staining.Results:?1?gross observation of the specimens:at the 12^th week,the femoral condyle defect of RGDmx group was completely filled with regenerated bone tissues and healed well with the surrounding bone tissue.RADA16-?group had a slight introcession in the central part of regenerated bone tissues.In PBS group,the regenerated bone tissue was few,and the central part of its bone defect still had a significant defect.?2?Micro-CT observation:The bone defect area was completely covered with regenerated bone tissues in RGDmx group,with complete bone remodeling;RADA16-?group also showed more new bone formations on bone defects,but its bone remodeling level was lower than RGDmx group;although a great number of new bone tissues were formed in PBS group,there were still a small number of bone defects.?3?Micro-CT quantitative analysis:the BMD?bone mineral density?,BV/TV?bone volume/total volume?and Tb.N?Trabecular number?were quantitatively analyzed by Micro-CT.The parameters of the three experimental groups had statistical differences at 12 weeks after operation among each group.?4?Histological section:the results of HE staining were consistent with those of Micro-CT analysis.Conclusion:The new-type self-assembled polypeptide hydrogel RGDmx scaffolds can better repair the femoral condylar defects of SD rats than the traditional RADA16-?hydrogel.