Osteochondral tissue engineering based on biomimetic osteochondral scaffold contained calcified cartilage layer compounding with ADSCs in vitro and vivo

摘要

Introduction: The biomimetic osteochondral scaffold contained calcified cartilage layer (CCL) was fabricated with slik fibroin (SF) and hydroxyapatite (HA). To investigate the effects of biomimetic osteochondral scaffold contained CCL compounding with ADSCs on regeneration of the osteochondral defect, especially the role of CCL in vivo, and explore the feasibility of this design as a concept of osteochondral tissue engineering. Materials and Methods: We fabricated a novel biomimetic osteochondral scaffold with CCL using SF and HA by the combination of paraffin-sphere leaching and modified temperature gradient-guided thermal-induced phase separation (TIPS) technique (Figure 1). ADSCs were seeded into the scaffold. The structure of scaffold, the cell adhesion, proliferation and viability on scaffold were observed by SEM, micro-CT, Dead/Live staining and other tests. Two different cell fluorescence labeling technique (DiO/Dil) were adopted for verifying the isolation of CCL between the osteogenic and chondral layers. The osteochondral defect model on rabbit bilateral knees were established, and implanted with the CCL+ADSCs, Non-CCL+ADSCs, CCL (no cells) and non-treated groups. At 4w, 8w and 12w after implantation, gross observation score, histological and immunohistochemical assessment, biochemical quantitative and biomectianical testing of new osteochondral tissue, micro-CT scans for new bone, were executed. Figure 1. Schematic diagrams of preparation process Results and Discussion: The scaffold had a consecutively overlapping trilayer structure with different densities and pore structures, approximately bionic the normal osteochondral structure. SEM, Dead/Live staining and other tests had shown good ability of cell adhesion, proliferation on scaffold. The CCL effectively isolated the cells among chondral and osteogenic layers(Figure 2). The cartilage regeneration in CCL+ADSCs group was better than Non-CCL+ADSCs group and CCL group, mainly reflected in flatness and integrity of cartilage. The content of GAG and type Ⅱ collagen in new cartilage tissue in CCL+ADSCs group more than the other groups(Figure 3). The results meant that the CCL contributed to the growth of cartilage. The quality and intensity of new bone in CCL+ADSCs and Non-CCL+ADSCs groups were higher than the CCL group, and no significant differences between first two groups. It meant that the ADSCs produced positive effects on the growth of bone. Figure 2. The isolated role of CCL Figure 3. Histological assessment of new osteochondral tissue Conclusion: The biomimetic osteochondral scaffold contained CCL approximately bionics the normal osteochondral structure, the CCL can isolate the different microenvironment for the growth of cartilage and bone. The scaffold compounding with ADSCs satisfactory regenerate the rabbit osteochondral defect, the CCL can accelerate the growth of cartilage tissue, especially the secretion of ECM, and the ADSCs contributes to the quality and intensity of new bone. the National Natural Science Foundation of China (Nos.31470937, 31300798, 31000432 and 81272046).