Research ArticleIMMUNOLOGY

Engineered probiotics biofilm enhances osseointegration via immunoregulation and anti-infection

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Science Advances  13 Nov 2020:
Vol. 6, no. 46, eaba5723
DOI: 10.1126/sciadv.aba5723
  • Fig. 1 Characterization and antibacterial performance of L. casei biofilm.

    (A) General concept of using inactivated L. casei biofilm to prevent MRSA infection and improve bone tissue regeneration simultaneously. (B) Surface morphology of L. casei biofilm–modified AHT-Ti. (C) Live/dead (green/red) staining of L. casei biofilm–modified AHT-Ti. (D) Section image of L. casei-AHT-Ti. (E) Growth of MRSA on the surface of Ti, AHT-Ti, and L. casei-AHT-Ti and corresponding photos of bacteria solution after 12 hours (right). Photo credit: L.T., Hubei University. (F) Number of MRSA colonies in the groups of Ti, AHT-Ti, L. casei-AHT-Ti-1 (1 day), L. casei-AHT-Ti-2 (2 days), and L. casei-AHT-Ti (3 days). (G) Lactic acid and (H) H2O2 detections from L. casei-AHT-Ti-1, L. casei-AHT-Ti-2, and L. casei-AHT-Ti with different culture time of biofilm. (I) SEM images of bacteria on the surface of samples. n = 3 independent experiments per group, *P < 0.05, **P < 0.01, and ***P < 0.001. ns, not significant.

  • Fig. 2 Activation of macrophages by L. casei-AHT-Ti.

    (A) Macrophages in the presence of Ti, AHT-Ti, and L. casei-AHT-Ti were stained by 4′,6-diamidino-2-phenylindole (DAPI; nucleus) and FITC-conjugated phalloidin (F-actin). Scale bar, 50 μm. (B) Cell viability of macrophages. Gene expression of (C) TNF-α, (D) OSM, and (E) IL-10. (F) Flow cytometry results of CD11c (M1 marker) and CD206 (M2 marker) expression (gated on CD11b+) after 2 and 3 days of treatments. (G) Venn diagram of differentially expressed genes treated by Ti and L. casei-AHT-Ti (top) and PCA plot shows the obvious separation gene expression of macrophages (bottom). (H) Up-regulated GO 514 enrichment analysis in L. casei-AHT-Ti compared with Ti. FDR, false discovery rate; JAK, Janus kinase; STAT, signal transducer and activator of transcription. (I) KEGG pathway analysis of the identified different gene expression. MAPK, mitogen-activated protein kinase. (J) Gene expression of TNF-α, OSM, and IL-10 in the presence of TLR2 inhibitor. n = 3 independent experiments per group, *P < 0.05 and **P < 0.01.

  • Fig. 3 Generation of osteoinductive cytokines by macrophages.

    (A) Expression change of the singled-out chemokines, cytokines, and growth factors between Ti and L. casei-AHT-Ti. (B) Expression of genes related to the production of OSM including CD14 (lipopolysaccharide receptor), COX2, and EPR. (C) Protein levels of CD14, COX2, PGE2, EPR, OSM, IL-6, TNF-α, IL-1β, and IL-10 were further quantified by ELISA assay. (D) Complete process of macrophage activated by L. casei-AHT-Ti for further stimulating osteogenesis. n = 3 independent experiments per group, Ti versus L. casei-AHT-Ti, *P < 0.05, **P < 0.01, and ***P < 0.001. IFN, interferon.

  • Fig. 4 Osteogenic differentiation of MSCs.

    (A) Cell morphology of MSCs in the presence of Ti, AHT-Ti, and L. casei-AHT-Ti. Scale bar, 50 μm. (B) Cell viability of MSCs. (C) ALP activity of MSCs in the groups of Ti, AHT-Ti, and L. casei-AHT-Ti after 14 days. (D) Osteogenic-related gene expression of ALP, RUNX2, and OCN after 14 days. (E) Alizarin red staining of samples after 14 days. Scale bars, 200 μm. Photo credit: L.T., Hubei University. (F) Quantification of the Alizarin red staining intensity. n = 3 independent experiments per group, *P < 0.05 and **P < 0.01.

  • Fig. 5 Bone integration of L. casei-AHT-Ti in vivo.

    (A) H&E and Giemsa staining of bone tissue around the implants. (B) Remaining bacteria colonies of the Ti, AHT-Ti, and L. casei-AHT-Ti rods pulled out from the bone tissues. Photo credit: L.T., Hubei University. (C) Antibacterial efficiency of L. casei-AHT-Ti compared with Ti and AHT-Ti groups in vivo. (D) Micro-CT results. 3D, three-dimensional. (E) BV/TV values of Ti, AHT-Ti, and L. casei-AHT-Ti. (F) Van Gieson’s picrofuchsin staining of the newly formed bone tissues at the bone-implant interface. (G) Bone area ratios of samples calculated from the Van Gieson’s picrofuchsin staining. n = 3 independent experiments per group, **P < 0.01 and ***P < 0.001.

Supplementary Materials

  • Supplementary Materials

    Engineered probiotics biofilm enhances osseointegration via immunoregulation and anti-infection

    Lei Tan, Jieni Fu, Fan Feng, Xiangmei Liu, Zhenduo Cui, Bo Li, Yong Han, Yufeng Zheng, Kelvin Wai Kwok Yeung, Zhaoyang Li, Shengli Zhu, Yanqin Liang, Xiaobo Feng, Xianbao Wang, Shuilin Wu

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