•A non-repetitive domain and its multimers of silk fibroin were expressed by E. coli.•The corresponding target polypeptides F(1), F(4) and F(8) were cleaved clearly.•Their molecular weights are consistent with the predicted values.•Their amino acid compositions are similar to the predicted sequences.•The target polypeptides exhibit good hydrophilia and a negative ζ-potential.Silk fibroin heavy chain is the major protein component of Bombyx mori silk fibroin and is composed of 12 repetitive and 11 non-repetitive regions, with the non-repetitive domain consisting of a hydrophilic polypeptide chain. In order to determine the biomedical function of the non-repetitive domain or potentially use it to modify hydrophobic biomaterials, high-purity isolation is necessary. Previously, we cloned and extended a gene motif (f(1)) encoding the non-repetitive domain. Here, this motif and its multimers are inserted into a glutathione S-transferase (GST)-tagged fusion-protein expression vector. Motif f(1) and multimers f(4) and f(8) were expressed in Escherichia coli BL21 cells following isopropyl β-D-1-thiogalactopyranoside induction, purified by GST-affinity chromatography, and single bands of purified fusion proteins GST-F(1), GST-F(4), and GST-F(8), were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Target polypeptides F(1), F(4), and F(8), were cleaved clearly from the GST-fusion tag following thrombin digestion. Mass spectrometry results indicate that the molecular weights associated with fusion proteins GST-F(1), GST-F(4), and GST-F(8) are 31.5, 43.8, and 59.0 kDa, respectively, and with the cleaved polypeptides F(1), F(4), and F(8) are 4.8, 16.8, and 32.8 kDa, respectively. The F(1), F(4), and F(8) polypeptide chains are negatively charged with isoelectric points (pI) of 3.3, 3.2, and 3.0, respectively. The molecular weight and pI values of the polypeptide chains are consistent with the predicted values and the amino acid compositions similar to predicted sequences. FTIR and CD results show the molecular conformation of F(1) was mainly random coil, and more stable α-helix structure formed in longer molecular chain.

•A PEG-DE cross-linked small caliber porous silk fibroin tubular scaffold (SFTS)•PEG-DE cross-linked SF film had no inhibitory effect on DNA replication of cells.•Cells cultured on the SFTS showed good morphology, cell viability and proliferative activity.•SFTS would be beneficial to endothelialization.•SFTS had good suture retention strength and flexibility.Regenerated silk fibroin (SF) materials are increasingly used for tissue engineering applications. In order to explore the feasibility of a novel biomimetic silk fibroin tubular scaffold (SFTS) crosslinked by poly(ethylene glycol) diglycidyl ether (PEG-DE), biocompatibility with cells was evaluated. The novel biomimetic design of the SFTS consisted of three distinct layers: a regenerated SF intima, a silk braided media and a regenerated SF adventitia. The SFTS exhibited even silk fibroin penetration throughout the braid, forming a porous layered tube with superior mechanical, permeable and cell adhesion properties that are beneficial to vascular regeneration. Cytotoxicity and cell compatibility were tested on L929 cells and human umbilical vein endothelial cells (EA.hy926). DNA content analysis, scanning electron and confocal microscopies and MTT assay showed no inhibitory effects on DNA replication. Cell morphology, viability and proliferation were good for L929 cells, and satisfactory for EA.hy926 cells. Furthermore, the suture retention strength of the SFTS was about 23 N and the Young's modulus was 0.2–0.3 MPa. Collectively, these data demonstrate that PEG-DE crosslinked SFTS possesses the appropriate cytocompatibility and mechanical properties for use as vascular scaffolds as an alternative to vascular autografts.

The study aimed to investigate the adhesion, morphology and proliferation of Sprague Dawley (SD) albino rat bone marrow-derived mesenchymal stem cells (BMSCs) by inverted microscope, cell counting, MTT test and laser scanning confocal microscop (LSCM). On the regenerated A yamamai SF film or blend films, the cell morphology was almost the same as that on collagen (collagen type I) film, the cell adhesion rate was higher than that on plastic cell plate and B mori SF film after 1 h (p < 0.01) of culture, and the cell proliferation was significantly higher than that on collagen film (p < 0.01) and plastic cell plate (p < 0.01), and also obviously better than that on B mori SF film. On the other hand, the viability of BMSCs in the regenerated A yamamai SF porous scaffold was better than that in B mori SF porous scaffold and medical grade polyvinyl alcohol (PVA) sponge.

•A direct thrombin inhibitor hirudin was used to modify silk fibroin.•Antithrombogenic property of Hir-modified silk fibroin films was improved.•Hir-modified silk fibroin films supported adhesion and proliferation of HUVECs and HAVSMCs.•Proliferation of HAVSMCs on silk fibroin films was inhibited by increasing Hir concentration.Thrombus formation remains a particular challenge for small-diameter vascular grafts. In this study, the direct thrombin inhibitor hirudin (Hir) was used to modify silk fibroin films in an attempt to enhance its antithrombogenic properties. Hir was successfully attached to silk fibroin and uniformly distributed in the regenerative material. Hir-modified films showed good cytocompatibility, and supported adhesion and proliferation of fibroblasts (L929), human umbilical vascular endothelial cells (HUVECs) and human aortic smooth muscle cells (HASMCs). Proliferation of HAVSMCs was inhibited by increasing Hir concentration. Activated partial thrombin time (APTT), prothrombin time (PT) and thrombin time (TT) of Hir-modified silk fibroin tubular scaffolds (SFTSs) were all increased markedly compared with fresh rabbit blood, ethanol-treated SFTS and unmodified SFTS, demonstrating the improved antithrombogenicity of SFTSs following modification with Hir.