Biological Safety And Biological Activity Of Designer Self-assembling Peptide Nanofiber Hydrogel Scaffolds For Bone Repair

PART 1 PREPARATION AND CHARACTERIZATION OF DESIGNER SELF-ASSEMBLING PEPTIDE NANOFIBER SCAFFOLDSObjectiveTo design and synthesize a self-assembling peptide(SAP),D-RADA16,which was only made of D-amino acids.We initially confirmed the characterization of SAP L-RADA16 and D-RADA16 by circular dichroism(CD)spectroscopy,transmission electron microscopy(TEM)and rheometer to investigate whether the two SAPs were enantiomers and possess the ability of self-assemble into interwoven nanofibers and study the mechanical properties of them.Then,we appraised the enzymatic stability of the SAPs with proteinase K.We want to develop a desinger biomaterial which show good biological activity and stability and can be synthesized commercially with high productivity for bone repair.MethodsThe SAP scaffolds L-RADA16 and D-RADA16 were custom-synthesized.And then,they were purified by high performance liquid chromatography(HPLC)and analyzed by mass spectrometer(MS).The secondary structure of the chiral RADA16 peptides were examined by CD measurements and the microstructure of those are explored by TEM.The rheological properties of the chiral SAP were analyzed with the rheology measurement.Having been degraded by the chiral SAPs,the protease K was used to examine the concentration of the intact peptide through HPLC.ResultsThe analysis of HPLC indicated that the purity of L-RADA16 and DRADA16 were 95.535 % and 95.9016 % respectively.The analysis of MS indicated that both of the relative molecular weights of l-RADA16 and DRADA16 were 1712.77.CD spectroscopy revealed that D-RADA16 has a nearly mirror image of L-RADA16 spectrum,suggesting that D-RADA16 being the chiral enantiomer of L-RADA16.TEM morphological studies denoted that D-RADA16 indeed formed well-ordered nanofibers in aqueous solution,and the diameters of nanofibers assembled from L-RADA16 and D-RADA16 were 16.34 ± 6.13 and 13.52 ± 2.94 nm,respectively.When D-RADA16 was at a concentration of 5 mg/ml,the storage modulus(G')values over the entire frequency range exceeded those of the loss modulus(G''),reflected the gel-like property.In contrast,the G' and G'' values obtained from the measurement with the D-RADA16 at a concentration of 2.5 mg/ml,both were low,which demonstrated a liquid-like property.The results were quite similar to that of L-RADA16.About 75 % and 60 % of D-RADA16 remained after 2 and 6 h of incubation with proteinase K,while less than 25 % and 8 % of L-RADA16 remained;more than 50 % of D-RADA16 remained,while less than 6.5 % of L-RADA16 remained after 24 h.The results illustrate that D-RADA16 exhibits stronger resistance than L-RADA16 to proteinase K digestion.ConclusionThe result denotes that D-RADA16 and L-RADA16 are enantiomers in molecular arrangement and secondary structure.Besides,D-RADA16 can form well-ordered nanofiber scaffolds in aqueous solution.Moreover,At a relatively higher concentration(5 mg/ml),the chiral SAP reflected the gel-like property in aqueous solutions,while at a relatively lower concentration(2.5 mg/ml),the chiral SAP reflected the liquid-like property in aqueous solutions.The result demonstrates that D-RADA16 and L-RADA16 are essentially similar to the mechanical properties.Lastly,D-RADA16 exhibits stronger resistance to proteinase K digestion compared with its chiral enantiomer of L-RADA16.PART 2 BIOCOMPATIBILITY AND BIOLOGICAL SAFETY OF DESIGNER SELF-ASSEMBLING PEPTIDE NANOFIBER HYDROGEL SCAFFOLDSObjectiveTo evaluate the biocompatibility and biological safety of designer SAP nanofiber hydrogel scaffolds,hoping to provide experimental evidence for the clinical application of the material.MethodsAccording to biological evaluation of medical devices(GB/T 16886.1-2011/ISO 10993-1:2009),physiological saline lixivium of the biomaterial or the hydrogel biomaterial was used in vitro cytotoxicity test,themolysis test,acute toxicity test,pyrogen test,intradermal irritation test,and micronucleus test to evaluate the biocompatibility of the material.ResultsIn the cytotoxicity test,the relative growth rate of the experimental group was above 90 %,and the toxicity of D-RADA16 was graded 1,indicating no cytotoxicity.The hemolysis rate of the lixivium was 0.96 %,which was lower than the national standard(< 5 %).The activity of mice was normal in the acute toxicity test.No animal died and no toxicity symptom or adverse effect was shown within 7 d.The increase of average daily weight among the experimental group and control group showed no statistically significant difference(P>0.05)after 7 d.The mice were sacrificed after 7 d,and no abnormal changes were observed in general observation and HE staining of liver,spleen,and kidney.The maximum increase of body temperature in the experimental group was 0.5 oC,and the total increase of body temperature of the three experimental animals was 1.2 oC,which met the national standard in the pyrogen test(< 1.4 oC).Intradermal irritation test results showed that the primary irritation index were all 0 grade in every defined time point.In the micronucleus test,the results showed that there were no statistically significant differences between the experimental groups and the negative control group(P>0.05),and there were statistically significant differences between the experimental groups and the positive control group(P<0.01).Therefore,potential mutagenicity was not observed in the micronucleus test.ConclusionDesigner SAP hydrogel scaffolds D-RADA16 is non-cytotoxic,non-hemolytic,non-acute toxic,non-pyrogenic,non-skin irritation,nongenetic toxic,and has good biological safety and biocompatibility,which exhibits a promising clinical application prospect and provides theoretical basis for the further research of cell biology study and clinical application.PART 3 EFFECT OF DESIGNER SELF-ASSEMBLING PEPTIDES NANOFIBER HYDROGEL SCAFFOLDS ON BIOLOGICAL ACTIVITY OF RAT BONE MESENCHYMAL STEM CELLSObjectiveFirstly,rat bone mesenchymal stem cells(BMSCs)were isolated as the seed cells.Next,the cells formed three-dimensional culture system on the SAP nanofiber scaffolds.Lastly,we evaluate whether D-RADA16 formed a stable nanofiber scaffolds,providing a similar 3D microenvironment for the adhesion,proliferation,and migration of BMSCs just as L-RADA16.This study may have implications for the application of D-form SAP and provide preliminary experiment data for their in vivo study and clinical application in bone regeneration and repair.MethodsRat BMSCs were cultured with whole bone marrow method and the cells of third-passage or fourth-passage at sub-confluence were used for the following experiments.After the cells were incubated for 3,5 or 7 days,MTT assay was used to determine the extent of cell proliferation seeded on the chiral SAP hydrogels.In the case of migration test,cell culture transwell inserts were used for peptide hydrogelation,and BMSCs were seeded on top of the peptide gel in the inserts.After 7 days of co-culture,the migration of BMSCs into the hydrogel scaffolds was examined using calcein-AM staining.ResultsMTT assay demonstrated that there was no significant difference of the proliferation rates between the D-form and L-form SAP scaffolds after 3 days of culture(P>0.05).Peptide with low concentration of 1.25 or 2.5 mg/ml had no statistically significant difference of cell proliferation in comparison to the conventional 2D cell culture method(control)(P<0.05).In contrast,peptide with high concentration of 5 or 10 mg/ml showed statistically significant cell proliferation in comparison to the control(P>0.05).However,there was no statistical significance in cell proliferation rate when the peptide of 5 mg/ml was compared to a higher concentration of 10 mg/ml.Therefore,the best concentration of the chiral peptides for cell culture was 5 mg/ml,and the concentration was selected for further experiments.Subsequently,BMSCs were grown on the chiral scaffolds of 5 mg/ml and the control for 5 and 7 days.Significant difference of the chiral scaffolds compare to the control were noted after 5 days of culture(P<0.05).Nonetheless,there are no significant difference of the chiral scaffolds compare to the control after 7 days of culture(P>0.05).In the cell migration assay,the confocal microscopy 3D reconstruction images showed that cells migrated into the L-RADA16 and D-RADA16 scaffolds for ~285 ?m and ~186 ?m respectively after 7 days of co-culture.The result indicates that both of the two chiral peptides promote cell migration into the 3D nanofiber scaffolds.ConclusionDesigner SAP D-RADA16 scaffolds can provide a 3D culture model for cell culture.D-RADA16,as the enantiomer of L-RADA16,can provide a suitable microenvironment for BMSCs proliferation and migration,and exhibit a good clinical application prospect.