Enzymatic Grafting Of Chitooligosaccharide Onto Silk Fibroin And Preparation Of The Fibroin-based Biomimetic Mineralized Material

Silk fibroin(SF),with the characteristic of good mechanical behavior,moisture penetrability,biocompatibility and biodegradability,has the potential applications in the fields of tissue engineering and biomedical materials.In this paper,chitooligosaccharide(COS)was enzymatically grafted onto SF using laccase to prepare SF-g-COS membrane.Subsequently,the phosphorylation of COS in SF-g-COS membrane was carried out using hexokinase,and hydroxyapatite(HAp)was deposited on the surface of phosphorylated SF-g-COS membrane by in situ biomimetic mineralization,aiming at obtaining the fibroin-based biomimetic mineralized material and expanding the applications of silk fibroin-based biomaterials.P-Hydroxyphenylacetamide(PHAD),as the model compound of tyrosine residues in fibroins,was firstly used to investigate the efficiency of laccase catalyzed self-polymerization of PHAD and the reaction of grafted COS.The structures of the formed products were characterized and analyzed by means of Ultra performance liquid chromatography-Triple quadrupole detection(UPLC-TQD),Fourier transform infrared(FTIR)and ~1H-Nuclear magnetic resonance(~1H-NMR).Meanwhile,the efficacy of grafting COS in SF solution by laccase was explored by Size exclusion chromatography(SEC),Sodium dodecyl sulfate-Polyacrylamide gel electrophoresis(SDS-PAGE)and analysis of particle size.Besides,the antioxidant,antibacterial properties,thermal behavior and biocompatibility for the membrane samples were also compared.The results indicated that PHAD can be polymerized using laccase,and further grafted onto COS chains via the Schiff-base and Michael-addition reactions.The results from SDS-PAGE and SEC analysis evidently verified that the molecular weight of SF was increased,revealing the occurrence of self-crosslinking and grafting of COS onto fibroin molecules.As a result,satisfactory antioxidant and antibacterial properties for the SF-g-COS membranes were obtained.Based on the above-mentioned investigation,the phosphorylation of COS was carried out in the presence of hexokinase and adenosine triphosphate(ATP).Efficacies and mechanism of enzymatic phosphorylated COS were evaluated by the means of high-performance liquid chromatography(HPLC),matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF MS),FTIR and the content of phosphorus.The results indicated that hexokinase has the potential ability to convert COS unit to aminoglucose-6-phosphate and form adenosine diphosphate(ADP)simultaneously.The characteristic peak of P=O and C-O-P in phosphorylated COS appeared from the result of FT-IR.When the SF-g-COS membrane was phosphorylated,the content of phosphorus increased significantly.Furthermore,the phosphorylated SF-g-COS membrane was alternately mineralized in Ca/P solution,the deposition of mineral phase of HAp was analyzed by means of X-ray diffraction(XRD),Scanning electron microscopy(SEM),and Energy dispersive spectrometer-Mapping(EDS-Mapping).The results showed that the phosphorylated treatment can improve the mineralization effect of silk fibroin membranes,and more hydroxyapatite(HAp)mineral phases were formed on the surface of phosphorylated SF-g-COS membrane.Besides,the effects of proliferation and adhesion for the MG-63 osteoblast cells on the surface of SF were satisfactory,indicating that the newly-built fibroin-based material has good biocompatibility and exhibits the potential application prospects in the field of tissue engineering materials.