Abstract: BACKGROUND: To repair cartilage, cartilage calcification layer, and bone tissue at the same time, it is necessary to design a heterogeneous scaffold and add an intermediate layer that mimics the cartilage calcification layer of natural bone cartilage tissue. The three-layer structure of three-phase scaffold can reconstruct cartilage, calcified cartilage, and subchondral bone at the same time, so as to realize the integration of bone and cartilage repair.
OBJECTIVE: To construct silk fibroin/type II collagen/hydroxyapatite three-phase composite, and to study its physicochemical properties and biocompatibility. 
METHODS: Silk fibroin/type II collagen/hydroxyapatite three-phase composite was constructed by uneven sedimentation and low-temperature 3D printing. The porosity, water absorption expansion rate, and mechanical properties of the composite were detected. Mouse chondrocyte ADTC-5 was seeded on the surface of the scaffold. The distribution, adhesion, and extension of chondrocytes on the scaffold were observed under a scanning electron microscope. MTT assay was used to detect cell proliferation. Hematoxylin-eosin staining was used to observe the cell-scaffold composite morphology. 
RESULTS AND CONCLUSION: (1) The composite scaffold was malleable and viscoelastic, with a porosity of (97.7±1.5)%, a water absorption expansion rate of (1 410.95±66.55)%, and an elastic modulus of (35.3±2.5) kPa. Under the scanning electron microscope, the pore network structure of the composite could be seen, with good connectivity of each pore, large pore diameter of (210.5±20.5) μm, small pore diameter of (75.5±16.5) μm, porosity (97.7±1.5)%, and average wall thickness (92.5±18.5) μm. (2) After 3 days of culture, chondrocytes adhered to the internal pores of the scaffold and secreted a large amount of extracellular matrix under the scanning electron microscope, and the cell morphology was distinct. (3) MTT assay exhibited that with the extension of the culture time, the chondrocytes on the scaffold continued to proliferate. (4) Hematoxylin-eosin staining demonstrated that after 3 days of co-culture, chondrocytes grew adherently along the internal voids of the scaffold material, with a fusiform shape, dispersed distribution and a small number. After 14 days of co-culture, the number of cells increased significantly. Cells grew like a colony, and the connections between cells could be clearly seen. (5) These findings verify that silk fibroin/type II collagen/hydroxyapatite composite prepared by uneven sedimentation technology has good physical and chemical properties and biocompatibility. 

Key words: three-phase composite, uneven, sedimentation, printing, construction, physical and chemical properties, biocompatibility