To Investigate A Biomimetic Three-layered Silk Fibroin/β-T CP Scaffold

ObjectiveTo evaluate the biological safety of a novel scaffold made by silk fibroin and β-TCP. The scaffolds mixed with rat articular chondrocytes were used to construct a tissue-engineered cartilage.Methods(1) The scaffold was prepared by the improved commixing and freeze-drying method. It consists of silk fibroin and β-TCP from the upper to lower layer respectively, and the intermediate layer was made by mixed silk fibroin (40%) and β-TCP (60%).(2) The scaffolds were assessed via macroscopic observation, scanning electron microscope. And then the pore diameter and porosity was observed.(3) The biological safety experiments included system toxicity test, sub-acute toxicity test, blood compatibility test, CCK-8test, H&E staining.(4) The second generation rat articular chondrocytes were inoculated on to the silk fibroin of the scaffolds in vitro by negative pressure suction technique. The seeding efficiency and adhesive rate of seed cells were calculated after seeding. Four weeks after in vitro culture, the tissue-engineered cartilage of the scaffolds were assessed via macroscopic observation, scanning electron microscope, safranin O and alcian blue staining.Results(1) The diameter and the height of the scaffold were4mm and10mm, respectively. The pore diameter and porosity of the different parts:the upper layer was150-200μm and91.5%±2.2%, the intermediate layer was30-50μm and74.2%±3.8%, the lower layer was400-500μm and70.6%±4.2%.(2) After injection and implantation of extracts and scaffolds, general condition of animals remained normal. Weight, red and white blood cell of experimental animals altered slightly after two weeks. H&E of heart, liver and kidney remained normal. Hemolytic rate of the scaffold is2.09%, which met the requirement of hemolysis test standard (<5%) as a medical material. CCK-8test showed that the scaffolds did not induce any cytotoxicity.(3) The seeding efficiency and adhesive rate of the biomimetic three-layered scaffold was81.8%and68.3%, respectively.(4) Safranin O and alcian blue staining showed that the chondrocytes have good adherent and proliferative ability in the scaffold.ConclusionThe silk fibroin/β-TCP scaffold has good biological safety, and the biomimetic three-layered scaffold applies to tissue engineering.