| Tissue engineering appeared in the 1980 s, is an emerging discipline in the field of biomedical engineering. It provides a new treatment for the repair of body tissues or organs defected by disease, trauma etc. Tissue engineering including three basic elements:seed cells, artificial scaffolds and growth factor. Scaffold material plays an important role in the field of tissue engineering. An ideal scaffold should mimic natural extracellular matrix as much as possible, for providing a good growth environment for cells. Basically, they should have some required performances, such as good biocompatibility, good mechanical properties, appropriate degradation rate and the three dimensional space of the porous structure, etc. Silk Fibroin(SF), a kind of natural protein fiber extracted from cocoon, having good biological compatibility, excellent mechanical properties, no antigenicity and easy processing, now has been made into a variety of shapes of scaffold and being studied widely in the field of tissue engineering. Gelatin(G) is a kind of collagen denaturation derivatives, having low antigenicity, good plasticity, but it’s mechanical properties is not strong enough. Composite scaffold is the hot spot in the research of tissue engineering scaffolds, since a single material have drawbacks more or less, combining two or more materials together can make up for each other, and to create a more excellent scaffold. Freeze-drying Method is a mature method which commonly used for manufacturing tissue engineering scaffolds, its advantage lies in making the scaffolds have three-dimensional porous structure, and the operation process is simple, mild and controllable.In this study, SF/G three-dimensional porous composite scaffolds with different mass ratio (7:3, 5:5,3:7) was prepared by freeze-drying technology under different pre-freezing temperature (-20 ℃,-80℃), using scanning electron microscopy (SEM) to observe the microscopic morphology of SF/G scaffold, X-ray diffraction (XRD) to determine the crystalline structure, thermogravimetric analyzer (TGA) to test its thermal stability, water absorption performance and porosity are also measured. Finally, picking out relatively appropriate mass ratio of SF/G composite scaffold and better pre-freezing temperature, then the periodontal ligament cells (hPDLSCs) was vaccinated on the scaffold which exhibit best comprehensive performance, the control group (hPDLSCs without scaffold) was setted at the same time, in order to test its biocompatibility(determined by MTT, cell electron microscope). Synthesize the above results, to evaluate the potential of SF/G three-dimensional porous composite scaffolds prepared by freeze-drying method in the field of tissue engineering application.Scanning electron microscopy (SEM) results show that the fibroin/gelatin composite scaffolds with mass ratio of 7:3 and 5:5 are better than 3:7 scaffolds in terms of pore uniformity, structural integrity and mutual connectivity between pores; At the same time, pore structure obtained under-80℃ pre-freezing temperature is also better than pore structure made under -20℃. X-ray diffraction (XRD) results show that the diffraction peak of SF/G composite scaffolds is near the 20.6°, which means the structure of fibroin was transformed into a more stable β-sheets structure. Thermogravimetric analysis (TGA) results show that the thermal decomposition temperature of SF/G composite scaffolds increase from 280° and 275° of single material to 300° of composite material, which means better thermal stability was obtained. Water absorption of SF/G composite scaffolds increase with the increase of gelatin proportion, but there exists a critical value, Water absorption will decrease above that; At the same time,-80℃ pre-freezing temperature is more beneficial for SF/G scaffolds ot obtain a higher water absorption than -20℃ pre-freezing temperature. Porosity results show that the scaffolds porosity decrease with the increase of gelatin ratio; at the same time, scaffold made under -80℃ pre-freezing temperature condition is easier to form the high porosity than -20℃ pre-freezing temperature. Biocompatibility results show that the 3th,5th,7th days OD values of the human periodontal ligament stem cells (hPDLSCs) that were inoculated on the SF/G composite scaffolds, are all higher than the control group; SEM figures of 5th and 7th days show that the human periodontal ligament stem cells (hPDLSCs) grew well on the SF/G scaffolds, cell robust proliferation, interconnected together and almost cover the whole surface of scaffold.Comprehensive the above results, the SF/G composite scaffolds with mass ratio of 7:3 and prepared under -80℃ pre-freezing temperature not only have the best physical and chemical properties, but also have good biocompatibility which means no toxic effects on cells. it is a suitable micro-environment for cell growth, and has certain potential in the field of tissue engineering application. |
