A Research On The Performance Of SF/COL/P(LLA-CL) Electrospun Three-dimensional Nanofiber Scaffold

Preparing nano fiber scaffold by using electro-spinning technique has been a research focus for recent years. The nano structure prepared by electro-spinning has the most similar properties to natural tissues. It’s has been extensively applied in the field of biomedical engineering due to its great biocompatibility, mechanical property and controllable porosity. The materials can be controlled in nano scale with high specific surface area. This study was to compound SF, COL and P(LLA-CL) by electro-spinning. Previous studies showed that the structural characteristics and chemo-physical abilities of SF are much similar to that of COL. They process great biocompatibility, non-toxicity; meanwhile COL is a major component of ECM, which can benefit the formation of natural bone. However, the applications on bone tissue engineering of both materials were constrained due to their inferior mechanical property and rapid degradation rate. P(LLA-CL) was used in this study. P(LLA-CL) is the copolymer of PLA and PLC, which processing great mechanical ability and biodegradability. Thus it can improve the disadvantages of both mentioned materials.Hence, in this study, the advantages of the SF/COL/P(LLA-CL) were used to synthesive a novel 3D nano scaffold.This study consisted of three components.1、To test the physiochemical ability of SF/COL/P(LLA-CL) scaffold. Based on the advantage of P(LLA-CL), HFIP was used as solvent to prepare scaffolds by using electro spinning technique (mass ratio was set as 0:100、30:70、50:50、 70:30、100:0). The preparation of the materials was finished by other group members previously. SEM, XRD DTA-TG were used to characterize the physiochemical property of the materials before and after crosslink. The results of SEM showed that the nano fibers were uniformly synthesized homogeneously distributed. XRD showed that molecule structure of SF/COL merely changed after crosslinking with copolymer. However β-sheet and crystallinity of the scaffolds increased to some extent compared with that od before crosslinking. Therefore, it can form a stable molecular conformation. Thermo stability showed a stable trend with the increase of P(LLA-CL) concentration. The contact angel of the material was greater than 90 degrees, demonstrating the hydrophobicity of the material. The addition of P(LLA-CL) has improved the hydrophilcity of the material, which can promote the adhesion, growth and proliferation of cells.2、To test the vitro biocompatibility of SF/COL/P(LLA-CL) scaffold. PDLSCs viability in the scaffolds was determined by MTT assay at specific time intervals of 1, 3,5, and7 days. Optical density was measured at 490 nm using amicroplate reader After 5,7,9 days in culture, the samples were sputter-coated with gold and observed by a SEM. Each sample of different mass ratio of the scaffold(lcmxlcm)was incubatedin sterile PBSbuffer salt solution at 37 ℃ for six weeks. At the end of each week, the residual of scaffold and PH values of each sample were measured for three times per week in order to take the mean value.As the results shows that the PDLSCs stick firmly to the surfaces of the membranes after crosslinking, grow well and secrete a great deal of matrices, which means the membranes can efficiently promote the cell adherence, spreading and multiplication. Our results also indicated that it was higher cell adherence and multiplication in the group of 70%P(LLA-CL) than other groups, and there were no obvious differences between this group and the cell culture plates. The scaffold of pure P(LLA-CL)degraded30.33% and 65.25% after the first and six week respectively and the degradation rate of different scaffold composed of 70:30,50:50,30:70 decreased gradually. It was considered that the addition of SF/ COL and neutral buffer solution decreased the degradation of P (LLA-CL). The result of measured PH between 6.51 and 7.03 means the degradation products of material is slightly acidic and close to the PH value of body tissue which is between 7.35 and 7.45. SF, COL and P (LLA-CL) are polymers, the degradation products, including carbon dioxide and water, isharmlessto the body and can be absorbed and metabolized by the body.3、Animal experiments to test the vivo biocompatibility. The scaffold sample was prepared before implantation as described in the in vitro cytocompatibility experiment.The concentration of rh.BMP-2 in culture medium is100ng/ml. After the PDLSCs were seeded and cultured on scaffolds for lweek in vitro, all of the scaffolds were implanted into subcutaneous pockets of nude mice. The animals were sacrificed after 4,8weeks implantation and the scaffold were removed for subcutaneous tissues. The experiment results in vivo of mice show that a great quantity of tissue grew into the blending composite nanofibers with P(LLA-CL) content of 70% and a large number of multinucleated giant cells could be seen around the nanofibers(Fig.12). The results shown thatthe 70%SF/COLP(LLA-CL)has good biocompatibility and certain potential of inductive osteogenesis.Above all, nanofibers has good pore structure and contribute to cell migration and nutrients dispered.This study aimed to prepare SF/COL/P(LLA-CL) blending composite nanofibers scaffolds using electrospinning technique, to improve the physical and chemical properties through the P(LLA-CL) joined and to assess the effect of glutaraldehyde crosslinked.Theblending composite nanofibers were inoculated with periodontal ligament stem cells(PDLSCs) in vitro. The cells grew well on the surface of materials, and had fine adhesionandproliferation. The cytocompatibilityand mechanical properties with P(LLA-CL)content of 70% are superior to other groups, which suggests the group is expected to become a new type of periodontal tissue engineering scaffolds.