Fig 1: The evaluation of osteogenic activity in Zn + CS/pPRL implants in vitro. (A) MC3T3-E1 cells were cultured with 25% extract of Zn implants or Zn + CS/pPRL implants for 24 h, and the cytoskeleton was stained with FITC labeled phalloidin. (B) the expression of ZIP4 mRNA in MC3T3-E1 cells treated with 25% extract of Zn implants or Zn + CS/pPRL implants for 48 h. (C) Representative image of FluoZin-3-stained MC3T3-E1 cells and fluorescence quantification analysis of intracellular Zn2+ concentrations. (D–E) MC3T3-E1 cells were cultured with 25% extract of Zn implants loaded with CS or/and PRL for 14 d. The cell activity and mineralization of MC3T3-E1 cells were analyzed using alizarin red S staining and ALP staining. Bars represent mean ± S.D. *p < 0.05; **p < 0.01
Fig 2: Effects of Zn + CS/pPRL implants on bone formation in rats with bone defects. (A) TRAP/ALP staining images of femurs with bone defects from rats. Purple represents TRAP positive; brown represents ALP positive. Scale bars, 100 μm. (B) BALP levels in serum detected using an ELISA assay. (C) Representative fluorescence images of femurs with bone defects from rats after xylenol orange (red) and calcein labelling (green). Dynamic histomorphometric analyses of femur including mineral apposition rate (MAR). Bars represent mean ± S.D. *p < 0.05; **p < 0.01
Fig 3: The promotional mechanism of Zn + CS/pPRL implants on bone formation in rats with bone defects. (A) Representative image of FluoZin-3-stained femur with bone defects. (B)The fluorescence quantification analysis of Zn2+ concentrations. (C) The serum concentrations of Zn2+ detected using ICP. (D) The expression of ZIP4 mRNA in femur with bone defects was detected using RT-qPCR (n = 3). (E) The expression of Runx2, Alpl, Osterix mRNA in femur with bone defects were detected by RT-qPCR (n = 3). Bars represent mean ± S.D. *p < 0.05; **p < 0.01
Fig 4: Analysis of the mechanism of cytotoxicity induced by excessive Zn2+. (A) MC3T3-E1 cells were incubated with cell medium containing different concentrations of Zn2+ for 24 h, and cell counting kit-8 (CCK-8) assay was used to detect cell viability. (B) Top: apoptotic cells were detected using a Terminal dUTP nick-end labeling (TUNEL) assay. Bottom: intracellular Zn2+ concentrations in MC3T3-E1 were detected using a Zn2+ fluorescence probe (FluoZin-3, AM). (C) The quantification analysis of positive TUNEL cells. (D) The fluorescence quantification analysis of intracellular Zn2+ concentrations. (E) RNA transcriptome sequencing analysis of MC3T3-E1 cells treated with excessive Zn2+ (180 µM) or control (n = 3). Heatmap of top-100 highly variable genes. (F) GO enrichment analysis results. (G–H) The expression Zip1-Zip14 mRNA and genes associated with osteogenesis (Runx2, Alpl, Osterix mRNA) in control cells or MC3T3-E1 cells treated with excessive Zn2+ were detected using RT-qPCR (n = 3). Bars represent mean ± S.D. *p < 0.05; **p < 0.01
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