Abstract: BACKGROUND: Among the biological materials developed by tissue engineering, gelatin, sodium alginate and 58S bioactive glass have good biocompatibility, suitable degradability and better osteogenic induction in bone defect repair.  
OBJECTIVE: To prepare gelatin/sodium alginate/58S bioactive glass scaffolds with three-dimensional printing technology and investigate their performance in vitro and biosafety.
METHODS:  Gelatin, sodium alginate and 58S bioactive glass were mixed with deionized water and stirred uniformly as printing ink. The scaffolds were prepared by three-dimensional printing technology and then lyophilized after cross-linking. (1) In vitro experiment: The morphological characteristics and compressive strength of the scaffolds were detected by scanning electron microscopy and universal material testing machine, respectively. The scaffolds were immersed into simulated body fluid for 16 weeks to observe the degradation rate. L929 cells were cultured with scaffold extract for 3 days, and the morphology and growth of cells were observed. The scaffolds were co-cultured with rat bone marrow mesenchymal stem cells for 0, 7, 14 and 21 days. The cell proliferation was detected by CCK-8 assay. The cell adhesion and survival were observed by DAPI staining. The expression of osteogenic related genes was detected by RT-PCR. (2) In vivo experiment: Full thickness bone defects with a diameter of 5 mm were made in the right mandibles of 10 SD rats; 5 in the experimental group were implanted with scaffolds, and 5 in the blank control group were not implanted with scaffolds. Liver and kidney function tests and histological staining of the liver, kidney, brain and bone defects were performed 4 weeks after surgery.  
RESULTS AND CONCLUSION: (1) In vitro experiment: Scanning electron microscopy showed that the surfaces of the scaffolds were rough and honeycombed. The average Young’s modulus of the scaffolds was 272.33 MPa. In the first 6 weeks, the scaffolds degraded rapidly and uniformly after soaking in the simulated body fluids. After the week 6, the degradation rate slowed down and remained roughly uniform, and the degradation rate reached 18% at the week 16. Inverted microscope showed that L929 cells grew well in the scaffold extract, and the morphology and structure of L929 cells were intact. With the extension of culture time, the proliferation rate of rat bone marrow mesenchymal stem cells increased. DAPI staining showed that the rat bone marrow mesenchymal stem cells adhered to the surfaces of the scaffolds and gradually crawled and expanded from initial accumulation to peripheral growth. RT-PCR assay revealed that the scaffolds could promote the mRNA expression of bone morphogenetic protein-2, osteocalcin and RUNX2 in rat bone marrow mesenchymal stem cells. (2) In vivo experiment: In the experimental group, the function of liver and kidney was not affected, and pathological damage of liver, kidney and brain tissue was not caused after implantation of scaffolds. Hematoxylin-eosin staining of the mandibular bone defect specimens indicated that the scaffolds of the experimental group were not completely degraded, and the new bone connected the host bone with the remaining scaffolds. A small amount of osteoblasts and inflammatory cells were observed around the new bone tissue, while a small amount of new bone and a large amount of fibrous tissue were seen at the edge of the host bone in the blank control group. (3) These results suggested that the three-dimensional printed gelatin/sodium alginate/58S bioactive glass scaffolds for bone defect repair possess favorable cytocompatibility, no obvious cytotoxicity or tissue toxicity, and have admirable biosafety.

Key words: three-dimensional printing, gelatin, sodium alginate, 58S bioactive glass, bone defect, scaffolds, cytotoxicity, biosafety