Abstract: Objective To assess the prospect of integrated biphasic silk fibroin scaffold made by annulus fibrosus-nucleus pulposus tissue engineering in application as integrated intervertebral disc（IVD） . Methods An integrated annulus fibrosus-nucleus pulposus（AF-NP）biphasic scaffold was made by silk fibroin using two different uncomplicated methods which were paraffin spheres-leaching method（outer AF phase）and phase separation method（inner NP phase） . The scaffold was investigated by general observation, stereomicroscope and scanning electron microscopy（SEM） . Its pore size, porosity, and compressive elastic modulus were determined. AF and NP cells were isolated from rabbit IVD and seeded into the corresponding phase of the scaffold respectively. The cell-scaffold complex was cultured for 48 hours. The biocompatibility of the scaffold was evaluated by SEM, live/dead staining while CCK- 8 assay was used to assess cell proliferation. Results Stereomicroscope and SEM showed that AF phase and NP phase integrated perfectly without cross-linking. Both phases possessed highly interconnected porous structure [pore size of AF and NP phase were（220.0±23.1） μm and（90.0±17.8） μm, respectively] and highly porosity（AF and NP phase were respectively 91% and 93%） . In addition, this silk biphasic scaffold had impressive mechanical properties（150.7±6.8） kPa. SEM revealed that disc cells attached to regions of pore walls, distributed uniformly and secreted extracellular matrix. Live/Dead staining and cell count kit-8（CCK-8）analysis showed that the silk composite scaffold was non-cytotoxic to disc cells. Conclusion This silk biphasic AF-NP scaffold has satisfied pore size, porosity, biomechanical properties and biocompatibility, so it is ideal candidate for IVD tissue engineering.

Key words: tissue engineering, silk, intervertebral disk, scaffold, annulus fibrosus, nucleus pulposus