Three-dimensional electrohydrodynamic printing of attapulgite/polycaprolactone scaffolds and osteogenic differentiation ability in vitro

doi:10.12307/2021.239

Abstract

Abstract: BACKGROUND: The polycaprolactone nanofiber scaffolds prepared by electrostatic spinning have a structure resembling extracellular matrix, but it is not conducive to cell adhesion, migration, proliferation and differentiation due to the poor hydrophilicity.
OBJECTIVE: To fabricate attapulgite/polycaprolactone scaffolds via three-dimensional (3D) electrohydrodynamic printing and evaluate the biological effects and osteogenic induction ability on mouse bone marrow mesenchymal stem cells.

METHODS: Attapulgite/polycaprolactone scaffolds with different concentration of attapulgite (0%, 0.5%, 1%, 2%) were prepared by 3D electrohydrodynamic, and the morphology and structure were observed by scanning electron microscope. The mouse bone marrow mesenchymal stem cells were co-cultured with four groups of scaffolds. The growth and morphology of bone marrow mesenchymal stem cells were observed with scanning electron microscope and Live/Dead staining at 7 and 2 days, respectively. The proliferation of bone marrow mesenchymal stem cells was detected by CCK-8 assay at 1, 3 and 5 days. The mRNA expression levels of osteogenic marker genes were detected by real-time PCR at 3, 7, and 14 days. Alkaline phosphatase activity was detected at 3, 7 and 10 days of co-culture.

RESULTS AND CONCLUSION: (1) Scanning electron microscopy results showed that four groups of scaffolds presented macropore morphology with the pore size of about 450 μm, and the surfaces of 0% and 0.5% attapulgite were smooth, while the scaffold surfaces in 1% and 2% attapulgite groups were rough. (2) Scanning electron microscopy showed that cells adhered and grew well on the surface of these scaffolds, and there were more cells grow in 1% attapulgite group than in other groups. (3) Live/Dead staining showed that cells adhered and grew well on the surface of four groups of scaffolds, among which 2% attapulgite group had the least cells. (4) The results of CCK-8 assay showed that the cell proliferation rate of 0.5% attapulgite group was higher than that of the other groups. (5) Real-time PCR showed that the expressions of alkaline phosphatase, osteocalcin, osteoblast-specific transcription factor and Runx-2 genes in 0.5% attapulgite group for 14 days were higher than those in the other groups. (6) The alkaline phosphatase activities of 0.5%, 1% and 2% attapulgite groups were all higher than 0% attapulgite group, in which the 0.5% attapulgite group was the highest at 3 and 7 days but 1% and 2% attapulgite groups were higher than other groups at 10 days of co-culture. (7) The 0.5% attapulgite scaffold has better biocompatibility and osteogenic induction potential.

Key words: bone, material, 3D electrohydrodynamic printing, attapulgite, polycaprolactone, bone marrow mesenchymal stem cells, biocompatibility, osteogenic differentiation, biomaterials, alkaline phosphatase activity

CLC Number:

Li Chenkai, Qin Wen, Liu Chun, Chen Wenyang, Zhao Hongbin. Three-dimensional electrohydrodynamic printing of attapulgite/polycaprolactone scaffolds and osteogenic differentiation ability in vitro[J]. Chinese Journal of Tissue Engineering Research, 2021, 25(34): 5459-5464.

Figures/Tables 6

2.4 各组支架对细胞成骨相关基因表达量的影响碱性磷酸酶：共培养3 d时，2%凹凸棒石组碱性磷酸酶基因表达量高于0.5%凹凸棒石组(P < 0.05)，其余组间两两比较差异均无显著性意义(P > 0.05)。共培养7 d时，1%凹凸棒石组高于其他3组(P < 0.05或P < 0.01)，其余组间两两比较差异均无显著性意义(P > 0.05)。共培养14 d时，0.5%凹凸棒石组高于0%，1%凹凸棒石组(P < 0.01或P < 0.05)，其余组间两两比较差异均无显著性意义(P > 0.05)，见图5A。 Ⅰ型胶原：共培养3 d时，1%凹凸棒石组Ⅰ型胶原基因表达量高于2%凹凸棒石组(P < 0.05)，其余组间两两比较差异均无显著性意义(P > 0.05)。共培养7 d时，0.5%凹凸棒石组高于其他3组(P < 0.01或P < 0.05)，其余组间两两比较差异均无显著性意义(P > 0.05)。共培养14 d时，0.5 % 凹凸棒石组高于其他3组(P < 0.01)，其余组间两两比较差异均无显著性意义(P > 0.05)，见图5B。 Osterix：共培养3 d时，0.5%凹凸棒石组Osterix基因表达量显著高于其他3组(P < 0.01)，其余组间两两比较差异均无显著性意义(P > 0.05)。共培养7 d时，0.5%凹凸棒石组高于0%，2%凹凸棒石组(P < 0.01，P < 0.05)，其余组间两两比较差异均无显著性意义(P > 0.05)。共培养14 d时，0.5%凹凸棒石组高于其他3组(P < 0.01)，其余组间两两比较差异均无显著性意义(P > 0.05)，见图5C。骨桥蛋白：共培养3 d时，0.5%凹凸棒石组骨桥蛋白基因表达量显著高于其他3组(P < 0.01或P < 0.05)，其余组间两两比较差异均无显著性意义(P > 0.05)。共培养7 d时，1%凹凸棒石组高于其他3组(P < 0.01)，其余组间两两比较差异均无显著性意义(P > 0.05)。共培养14 d时，0.5%凹凸棒石组高于0%，2%凹凸棒石组(P < 0.05)，其余组间两两比较差异均无显著性意义(P > 0.05)，见图5D。骨钙素：共培养3 d时，0.5%凹凸棒石组骨钙蛋白基因表达量高于其他3组(P < 0.01)，其余组间两两比较差异均无显著性意义(P > 0.05)。共培养7 d时，0.5%凹凸棒石组显著高于0%，2%凹凸棒石组(P < 0.05)，其余组间两两比较差异均无显著性意义(P > 0.05)。共培养14 d时，4组间两两比较差异均无显著性意义(P > 0.05)，见图5E。 Runx-2：共培养3，14 d时，4组间Runx-2基因表达量比较差异无显著性意义(P > 0.05)。共培养7 d时，0.5%凹凸棒石组高于0%，2%凹凸棒石组(P < 0.01)，1%凹凸棒石组高于0%，2%凹凸棒石组(P < 0.05)，0%和2%凹凸棒石组间相比较差异无显著性意义(P > 0.05)，见图5F。"

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