Sustained Release Effect Of Self-assembling Peptide Nanofiber Hydrogel Scaffold On TGF And The Study On It's Biological Characteristics

PART1 PREPARATION AND CHARACTERIZATION OF DESIGNER SELF-ASSEMBLING PEPTIDE NANOFIBER SCAFFOLDSObjectiveTo design and prepare new type of self-assembling peptides such as L-RADA16,D-RADA16 and L-RADA16-RGD which composed of several amino acids like Arg,Ala,Asp and so on.Circular dichroism,scanning electron microscope,transmission electron microscope and rheometer were used to detect the characterization of L-RADA16,D-RADA16 and L-RADA16-RGD.So that the microstructure could be realized to assess whether it can form a nano fiber scaffold.Also the mechanical properties and phisycal stability of the three SAP were compared.We hope to develop a bone repair material with nice stability and biological activity and can be batch produced.MethodsThe three SAP scaffolds were custom-synthesized and purified by high performance liquid chromatography(HPLC).The secondary structure of the L-RADA16?D-RADA16 and L-RADA16-RGD peptides were examined by CD measurements and the microstructure of those were explored by SEM and TEM.The rheological properties of the three SAP were analyzed with the rheology measurement.ResultsThe composition sequences of L-RADA16 and D-RADA16 are Ac-RADARADARADARADA-CONH2,which contain different L and D type of amino acids respectively.The amino acids have chiral symmetry and there is no difference besides that.The sequence of L-RADA16-RGD is Ac-RADARADARADARADARGDS-CONH2,which contains L type of amino acids.The analysis of HPLC revealed that the purity of L-RADA16,D-RADA16 and L-RADA16-RGD were 97.78%?97.45%and 96.68%respectively.CD spectroscopy indicated that all three SAP owned beta-sheet structure with double-peak,and L-RADA16 and D-RADA16 were nearly mirror symmetric which indicated they were enantiomer.TEM image showed that L-RADA16 and D-RADA16 can self assemble into ordered nano fibers in solution,the fibers diameter were 13.63±2.83 nm and 16.22±6.25 nm.However L-RADA16-RGD did not form a network structure effectively like the others and also with larger fibers diameter which was about 20.57±4.36 nm.Rheology assays showed at the concentration of 5 mg/ml,the storage modulus G' was greater than the loss modulus G",indicating that both L-RADA16 and D-RADA16 may become gel state and have some mechanical properties.There was no obvious difference between D-RADA16 and L-RADA16 in rheology.ConclusionThe result denotes that L-RADA16,D-RADA16 and L-RADA16-RGD are capable of forming a stable beta-sheet secondary structure.L-RADA16 and D-RADA16 can form an ordered nanofiber network scaffol in solution,while L-RADA16-RGD can only form a thicker and shorter fibrous filament with incompleted and unordered scaffold structure.L-RADA16 and D-RADA16 can form gels at the concentration of 5 mg/ml.These indicate that L-RADA16 and D-RADA16 have the similar physical and mechanical properties.PART 2 EFFECT OF SELF ASSEMBLING PEPTIDE NANOFIBER HYDROGEL SCAFFOLD ON TGF FOR CONTROLLED RELEASE AND FOR THE PROLIFERATION OF RAT BONE MESENCHYMAL STEM CELLSObjectiveTo evaluate the controlled release function and effect of L-RADA16,D-RADA16 and L-RADA16-RGD on TGF-beta 1.And SD rat bone mesenchymal stem cells(BMSCs)were obtained as seed cells and co-cultured with TGF+SAP nanofiber scaffolds to form a three-dimensional culture system.The proliferation promoting effect of L-RADA16,D-RADA16 and L-RADA16-RGD with TGF-beta 1 on BMSCs were compared to provide preliminary experimental basis for in-vivo study and clinical application of bone tissue regeneration.MethodsTGF-beta 1 was mixed with L-RAD A16,D-RADA16 and L-RADA16-RGD the three SAP solution respectively.PBS solution was added to make them self-assemble.40 ?L supernatant of the mixed solution was taken at the time point of 1 hours,2 hours,4 hours,8 hours,24 hours,72 hours and 168 hours respectively as the sample for test and 40?L PBS solution was added as a supplement.ELISA kit was used to detect the content of TGF-beta 1 in the samples.The results were calculated according to the 1-D unsteady state form of Fick's second law and the release curve was drawn to evaluate the controlled release effect.After that BMSCs were got from whole bone marrow of SD rat and cultured until it was steady to be subcultured.Cells of the 3rd or 4th generation with well growth were used for the following experiments.After the cells were co-cultured with these three SAP hydrogel scaffolds with TGF for 3 days,7 days and 14 days,CCK-8 kit was used to detect and evaluate the cell proliferation.ResultsTGF-beta 1 can be sustained and slowly released from L-RADA16,D-RADA16 and L-RADA16-RGD within 168 hours.There was a initial burst within the first 8 hours,while the rate slowed down reached the peak at 24 hours and a plateau occurred after that.The release rate of L-RADA16-RGD is higher than that of L-RADA16 and D-RADA16,and that of D-RADA16 was lower than the other two SAP.CCK-8 kit test results demonstrated that:in the 3rd days,proliferation promoting effect of L-RADA16 SAP containing TGF-beta 1 on BMSCs was better than L-RADA16 without that under the same concentration and the difference was statistically significant(P<0.05);at the same time cell proliferation effect on BMSCs in those groups with SAP co-cultured was statistically better,compared with the control group without anything(P<0.05).In the 7th day,the rate of cell proliferation in L-RADA16+TGF-beta 1 group was significantly better than that of D-RADA16+TGF-beta 1,L-RADA16-RGD +TGF-beta 1 and the normal two-dimensional culture control group(P<0.05).The results of the 14th days were similar to those in the 3rd day,and the proliferation rate of all the experimental groups was better than that of the control group(P<0.05).These results showed that the three kinds of SAP scaffolds could promote the proliferation of BMSCs,while SAP containing TGF-beta 1 had a better proliferation effect on BMSCs than the SAP scaffolds without that.ConclusionL-RADA16,D-RADA16 and L-RADA16-RGD,the new nano biological scaffold materials,can make a slow and sustained release of TGF-beta 1 within 168 hours.The delivery rate reached peak and then be a plateau after 8 hours.SAP is a nice material for controlled release of drug.besides,L-RADA16,D-RADA16 and L-RADA16-RGD,the new types of SAP scaffold materials can be used to construct a 3D co-culture system with cells.the SAP containing TGF-beta 1 could promote the proliferation of BMSCs in SD rats more effectively compared with normal SAP.SAP is a kind of material that can control drug release,which has a good prospect for clinical application.PART 3 EFFECT OF NEW SELF-ASSEMBLING PEPTIDE NANOFIBER HYDROGEL SCAFFOLD ON REGENERATION FOR BONE DEFECT OF RATSObjectiveTo evaluate the effect of D-RADA16 self-assembling peptide on bone regeneration,defect on femur condyles of SD rat were made as animal model in vivo.We hope for providing a preliminary experimental basis for further research in vivo and clinical application in bone tissue regeneration.MethodsBone defect of femoral condyle of rat were made and filled with self assembling peptide.Animal were killed after 4 weeks,8 weeks and 12 weeks and the femur specimens were taken for the observation of morphology.Growth of bone trabecular volume was assessed by Micro-CT and observed through HE staining.All these were used to analyze and evaluate the regeneration of bone defect.ResultsHealing difference of bone defect area between the experimental group with SAP and control group can be observed macroscopically.Micro-CT indicated that the D-RADA 16/nHA group and D-RADA 16 group had a more complete healing range compared with the control group,newborn bone tissue can be obviously seen in experimental groups.At 8 weeks,BV/TV,BMD and Tb.Th in the experimental groups were also higher than that of the control group,and statistical difference was significant(P<0.05).HE staining showed that nHA/D-RADA16 group and D-RADA16 group had more formed bone trabecular at the original defect interface,the defects almost disappeared and the number was significantly better than that of the control group.ConclusionThe D-RADA16 SAP scaffold material can promote the formation of bone trabecula of SD rats and the repair of bone defect in early time.SAP and nano hydroxyapatite can be combined to make an ideal filling material and has a good future for clinical application.