Fig. 5: In vitro and in vivo multiple protections against bacterial invasion.

a SEM images exhibiting the anti-biofilm effect against S. aureus and P. gingivalis on surfaces of CM dense layer and BC-g-PNCl (scale bars = 2 μm). b Quantitative measurements of antibacterial rates against S. aureus and P. gingivalis of BC-g-PNCl via CFU counting after samples are stored in artificial saliva for different duration times (n = 3 independent samples; error bars = SD; data are presented as mean values ± SD). c Anti-adhesion effect of CM dense layer, BC, BC-g-PAM, and BC-g-PNCl (n = 4 independent samples; ANOVA followed by Tukey’s multiple comparisons; ****adjusted P < 0.0001, adjusted P = 0.173 between BC-g-PAM and BC-g-PNCl; error bars = SD; data are presented as mean values ± SD). d, e Fluorescent images (scale bars = 200 μm) and SEM images (scale bars = 10 μm) exhibiting the anti-penetration effect against S. aureus of CM and BC-g-PNCl before (d) and after (e) simulated clinical immersion for 4 weeks, indicating penetrated bacteria on the bottom surface (green for live penetrated bacteria in fluorescent images, pseudo-yellow for penetrated bacteria in SEM images). f Schematic diagram showing the process of a short-term model of infected calvarial defects in rats to detect in vivo multiple protection against bacterial invasion. g Images of bacterial colonies formed by S. aureus survival on the surfaces of membranes (CM, BC, BC-g-PNCl, BC-g-PNCl/CS-HAP), soft tissues above the membranes, and bone substitutes below the membranes. h Reduction rates of S. aureus survival on the membrane surfaces, soft tissues above the membranes, and bone substitutes below the membranes, indicating the multiple protection effects of BC-g-PNCl and BC-g-PNCl/CS-HAP (n = 3 independent samples; ANOVA followed by Tukey’s multiple comparisons; *adjusted P = 0.0222, ****adjusted P < 0.0001; error bars = SD; data are presented as mean values ± SD). SEM observation was repeated three times independently, yielding similar results.