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. 2021 Jun 3;12:3303. doi: 10.1038/s41467-021-23069-0

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — a CCK-8 assay about the proliferation of MC3T3-E1 preosteoblasts cultured on Ti-OH and Ti-PAA-NCl after cells were cultured for 1, 3 and 7 days (n = 3; P = 0.673, 0.639 and 0.145 for 1, 3 and 7 days compared with Ti-OH, respectively; Student’s t test). b Morphology of MC3T3-E1 preosteoblasts cultured on Ti-OH and Ti-PAA-NCl for 1 day (green for F-actin, blue for cell nucleus, scale bar = 20 μm). c ALP activity of MC3T3-E1 preosteoblasts cultured on Ti-OH and Ti-PAA-NCl for 7 and 14 days (n = 3; P = 0.132 and 0.954 for 7 and 14 days compared with Ti-OH; Student’s t test). d Alizarin Red S staining of MC3T3-E1 preosteoblasts cultured on Ti-OH and Ti-PAA-NCl for 21 days (red for calcium nodules, scale bar = 1 mm) and semi-quantitative measurement of calcium content (n = 3; P = 0.147 compared with Ti-OH; Student’s t test). e Western blot results of osteogenic-related proteins (OCN, OPN and RUNX2) expressed in MC3T3-E1 preosteoblasts cultured on Ti-OH and Ti-PAA-NCl for 3, 7 and 14 days after osteogenic induction. f RT-qPCR results of expression levels of osteogenic-related genes (OCN, OPN and RUNX2) in MC3T3-E1 preosteoblasts cultured on Ti-OH and Ti-PAA-NCl for 3, 7 and 14 days after osteogenic induction (n = 3; P > 0.05 for all time points of these three genes between groups; Student’s t test). g In vivo biocompatibility. HE staining and CD68 immunofluorescent staining of tissues around Ti-OH and Ti-PAA-NCl embedded in the backs of nude mice for 4 weeks (fluorescent green for CD68, fluorescent blue for cell nucleus, white scale bars in HE images = 100 μm, black scale bars in HE images = 25 μm, scale bars in CD68 immunofluorescent images = 100 μm). All error bars = s.d.