Three-dimensional culture of chondrocytes/3D-printed composite scaffolds under compression loading / 中国组织工程研究

ABSTRACT

BACKGROUND: The silk fibroin/type II collagen composite scaffold has been prepared by low-temperature bio-3D printing technology in the previous study and the scaffold has good mechanical properties. Studies have shown that mechanical stimulation is beneficial to bone remodeling, and gradient loading strain is beneficial to the activation of osteoblasts and osteoclasts. OBJECTIVE: To co-culture silk fibroin/type II collagen composite scaffolds with chondrocytes under compression loading, to observe the proliferation of cells, and to observe the preliminary repair effect of silk fibroin/type II collagen composite scaffold on cartilage defects. METHODS: The silk fibroin/type II collagen composite scaffold was prepared by low-temperature 3D printing to detect the porosity of the scaffold. The passage 3 mouse chondrocytes ADTC-5 were inoculated on the silk fibroin/type II collagen composite scaffold and cultured under static culture and mechanical load respectively. (1) Static culture: blank scaffold was set as control, and cell proliferation was detected by MTT assay at 1, 3, 5, 7, 10, 14 days of inoculation. (2) Culture under mechanical load: blank scaffold was set as control. At 1 day after inoculation, 0％, 1％, 5％, 10％, 15％, 20％ compressive strains were applied to the cell-scaffold complex, and continued to load for 3 days. Cell proliferation was detected by MTT assay, and the distribution, adhesion and morphology of the cells on the scaffold were observed by scanning electron microscopy and hematoxylin-eosin staining. A cartilage defect of 3.5 mm in diameter was made in the bilateral knee joint of New Zealand rabbits. The silk fibroin/type II collagen composite scaffold was implanted onto the left side, and no material was implanted onto the right side. The repair site was observed at 8 weeks after surgery. RESULTS AND CONCLUSION: (1) The porosity of the scaffold was (89.3±3.26)％, which was conducive to cell attachment. (2) After 5 days of static culture, the chondrocytes proliferated well on the surface of the composite scaffold. Under 0％, 1％, 5％, 10％, 15％, 20％ compressive strains, the cell proliferation on the scaffold first increased and then decreased, wherein the cell proliferation was highest under 10％ compressive strain, and lowest under 20％ compressive strain. (4) Under the scanning electron microscopy, the chondrocytes in the 0％ load group were distributed in the surface of the scaffold with irregularities, the cell morphology was obvious, and the cell protrusions were fully extended. There were few or no chondrocytes on the contact surface of the 10％ load group, and more cells distributed on the lateral and internal surfaces of the first layer, but the cell morphology was flat with obvious protrusions. (5) Hematoxylin-eosin staining showed that the chondrocytes in the 0％ load group were concentrated on the surface of the scaffold, and there were almost no cells in the pores, while the chondrocytes in the 10％ load group were distributed in the scaffold pores. (6) There was still a circular defect model with no scaffold implantation, and no obvious repair appeared; similar hyaline cartilage appeared in the defect after scaffold implantation, but there was no adhesion to the surrounding defected cartilage, and the new hyaline cartilage was independent. Overall, the adsorption, proliferation and growth of chondrocytes on the silk fibroin-type II collagen scaffolds is better when the compressive strain is 10％, and the composite scaffold can be used as a repair material for cartilage defects.