Objective To prepare a spider silk protein bilayer small diameter vascular scaffold using electrospinning, and to observe the blood compatibility in vitro. Methods The Arg-Gly-Asp-recombinant spider silk protein (pNSR16), polycaprolactone (PCL), gelatin (Gt), and heparin (Hep) were blended. Spider silk protein bilayer small diameter vascular scaffold (experimental group) was prepared by electrospinning, with pNSR16 ∶ PCL ∶ Hep (5 ∶ 85 ∶ 10, W/W) hybrid electrospun solution as inner spinning solution and pNSR16 ∶ PCL ∶ Gt (5 ∶ 85 ∶ 10, W/W) hybrid electrospun solution as outer spinning solution, but pNSR16 ∶ PCL (5 ∶ 85, W/W) hybrid electrospun solution was used as inner spinning solution in control group. The scaffold structure of experimental group was observed under scanning electron microscope (SEM); and the hemolysis rate, recalcification clotting time, dynamic clotting time, platelet adhesion, and platelet activation in vitro were compared between 2 groups. Results SEM results showed that bilayer fibers of scaffold were quite different in experimental group; the diameter distribution of inner layer fibers was relatively uniform with small pores, however diameter difference of the outer layer fiber was relatively big with big pores. The contact angle, hemolysis rate, recalcification clotting time, and P-selectin expression of scaffold were (35 ± 3) ° , 1.2% ± 0.1%, (340 ± 11) s, and 0.412 ± 0.027 respectively in experimental group, and were (70 ± 4) ° , 1.9% ± 0.1%, (260 ± 16) s, and 0.678 ± 0.031 respectively in control group; significant difference were found in indexes between 2 groups (P lt; 0.05). With the extension of time, the curve of coagulation time in experimental group sloped downward slowly and had a long time; the blood clotting index values before 30 minutes were significantly higher than those in control group (P lt; 0.05). Platelet adhesion test showed that the scaffold surface almost had no platelet adhesion in experimental group. Conclusion The spider silk protein bilayer small diameter vascular scaffold could be prepared through electrospinning, and it has good blood compatibility in vitro.
To study how to repair the cartilage defect according to the principles of tissue engineering with acellular cartilage matrix as scaffold material. Methods The ear cartilage was obtained from a New Zealand white rabbit(weighing 2.4 kg )and then treated by a modified Courtman’s four-step method to produce the acellular cartilage matrix. Eighteen New Zealand white rabbits (aged 6 months, weighing 2.4-2.6 kg) with no sex l imit were divided into three groups. Forevery rabbit, two pieces of ear cartilage measured 1 cm × 1 cm were excised in each ear. Defects were repaired as follows: group A with the combined graft of acellular cartilage matrix and perichondium, group B with acellular cartilage matrix and group C with perichondium. Three animals in each group were killed 4 and 12 weeks postoperatively, respectively. Tissue samples obtained were analyzed with gross observation, hematoxyl in-eosin stain, Safranine O-alcian blue stain and type II collagen messenger RNA in situ hybridization respectively. Results In gross observation, the repaired sites in groups A and B were not change meaningfully in their shape 4 weeks postoperatively; but they felt a bit of thicker and harder 12 weeks postoperatively. In group C two repaired sites formed scabs at 2 weeks and perforated at 5 weeks. In histological observation, there was a sl ight inflammatory reaction surrounding the acellular cartilage matrix 4 weeks after it was implanted in groups A and B. The inflammatory cells were mainly lymphocytes. The perichondrium graft in group C was collapsed in the defects in HE stain. The defect sites were negative for Safranine O-alcian blue stain and type II collagen mRNA in situ hybridization in all groups. At 12 weeks cells were found in the acellular matrix which arranged in irregular manner in group A in HE stain and was positive for Safranine O-alcian blue stain and type II collagen mRNA in site hybridization. In groups B and C, no new cell was found in HE stain and the repaired sites were negative for Safranine O-alcian blue stain and type II collagen mRNA in situ hybridization. Conclusion Acellular