Construction of three-phase silk tissue-engineered ligament and preliminary analysis of its biocompatibility

SUN Li-guo; FAN Hong-bin; LI Hong-guo; QU Ling; CANG Bao-cheng; XUE Ying-sen; LIU Xin-cheng; LI Dan

doi:10.3969/j.issn.2095-252X.2015.11.004

Abstract：Objective To evaluate physicochemical properties and biocompatibility of the three-phase silk tissue-engineered ligament.Methods The silk mesh was woven, and silk ifbroin was extracted. The three-phase scaffold was modified with chondroitin sulfate, hyaluronic acid sodium, hydroxyapatite ( HA ) and silk fibroin. The surface morphology and element of the scaffold were determined. After bone marrow mesenchymal stem cells ( BMSCs ) seeding, the cell’s proliferation and adhesion were observed by scanning electron microscope ( SEM ) and laser confocal microscopy.Results After the removal of sericin, silk surface became smooth. The silk ifber diameter was ( 9.89±1.98 ) μm. After the removal of sericin, the diameter was ( 9.14±1.75 ) μm. No signiifcant differences existed in the diameter of silk ifber (P=0.221 ). A surface element analysis showed the Ca element in area C, S and Na elements in area B were all higher than those in area A. The pore size of the material was 60-80 μm. The cell adhesion rates of area A, B and C 4hrs later were ( 83.2±3.4 ) %, ( 81.8±2.8 ) % and ( 71.1±5.4 ) %, which were signiifcantly different from those 2hrs later (P=0.0001 ). It indicated that most of the cells were adherent to the material after 4hrs’ cells culture. One day after the seeding, the BMSCs in the material adhered well by SEM. Seven days later, the cells covered the material surface and the pseudopodia grown into internal were visible. Using life / dead cell staining, the cells cultured 7d were of higher proliferation than 1d by laser confocal microscopy. The cells were visible in multiple levels of the material. Conclusions The constructed three-phase silk tissue-engineered ligament has good biocompatibility, which is conducive to the proliferation of BMSCs. It lays the foundation for the vivo experiment in the next.