The Preparation And Application Of Oxidized Silk Fibroin Nanofibers

In this article,natural Bombyx mori silk and Antheraea pernyi silk(AS)were pretreated in fomic acid and then oxidized in sodium hypochlorite(NaClO)solutions.When the NaClO addition reached 10 mmol/g of protein,the final carboxyl concentration of oxidized silk was0.720 and 0.833 mmol/g of protein for BS and AS,where significantly increased form the initial carboxyl concentration of 0.293 and 0.347 mmol/g of protein for BS and AS,respectively.Thereafter,individual silk nanofibers(SNs were achieved after sonicating the oxidized silk slurries.The average diameter of resultant BS nanofibers was 105±27 nm with several microns length.A similar morphology was achieved in resultant AS nanofibers as well with the average diameter of 112±33 nm.Additionally,an aggregating-dispersing(redispersing)process using the SNs was strongly regulated by adjusting the pH.Thus,the negatively charged SNs could be concentrated to ~20 wt%.During this process,80.1±1.7 and 90.9±2.2 wt%(85.7±2.2 and93.6 ± 1.5 wt% for AS)of the BS aggregates was recovered at pH 5 and 3,respectively.Interestingly,the SN aggregates could be re-dispersing into water with slightly stir for 10 min at pH >7.Negatively charged carboxyl groups were also introduced onto regenerated silk fibroin(SF)solution with the oxidation by using NaClO.When the NaClO addition was 2 mmol/g protein,a final carboxyl content of 1.09 mmol/g SF was obtained,corresponding to ~47% of the primary hydroxymethyl groups on the silk.The oxidized silk fibroin(OxSF)aqueous solutions showed a strong tendency to self-assemble to nanofibrils at our condition.The nanofibrillar structure of OxSF was investigated by a Thioflavin T fluorescence method and TEM observation,after incubating the OxSF solution for a period of time.The assembly of OxSF followed a nucleation process where the resultant individual nanofibrils were characterized from the network nanofibril assembled by SF.The fractal studies revealed that the charge distribution of OxSF altered the assembly course and the morphology of the assembled nanofibrils.A higher surface charge caused more selective aggregation,which decreased the fractal dimension(Df)values for OxSF nanofibrils.The average diameter of resultant OxSF nanofibers was 17±3 nm with the Df value below 2.Nanoindentation measurements demonstrated the reinforcement of the hardness and modulus in OxSF-chitosan composite materials obtained by layer-by-layer deposition.Interestingly,both infrared spectroscopy and circular dichroism spectra demonstrated that the resulting oxidized silk(OxSF)self-assembled into ?-sheet structures in aqueous conditions and this contributed to the mechanical properties of the as-prepared silk-based scaffolds and the mineralized OxSF scaffolds(M-OxSF).The OxSF scaffolds had a compressive modulus of 53.6±11.8 MPa in the dry state,100 times higher than that of the SF scaffolds(35.5±12.2 kPa).After mineralization,the modulus of M-OxSF scaffolds was 758±189 KPa,while the modulus of M-SF scaffolds was 21 ± 8 kPa.Human bone marrow-derived mesenchymal stem cells(hMSCs)grown on the scaffolds during osteogenesis showed that the OxSF scaffolds supported the proliferation and differentiation of hMSCs in vitro.The results were investigated by Calcein AM staining and osteoblast marker expression seeded with hMSCs under differentiation conditions(gene expression profiles for ALP,Col1a1,OPN,and OC).