Research ArticleAPPLIED SCIENCES AND ENGINEERING

Lysostaphin and BMP-2 co-delivery reduces S. aureus infection and regenerates critical-sized segmental bone defects

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Science Advances  17 May 2019:
Vol. 5, no. 5, eaaw1228
DOI: 10.1126/sciadv.aaw1228
  • Fig. 1 Lysostaphin and BMP-2 co-delivery to a critical-sized segmental bone defect.

    A 2.5-mm segment of the radius is removed to create a critical-sized bone defect that does not spontaneously heal. A PEG-4MAL hydrogel functionalized with the adhesive ligand GFOGER and loaded with lysostaphin and BMP-2 is synthesized with S. aureus. Lysostaphin enzymatically creates holes in the bacterial cell wall, leading to lysis. These infected hydrogel scaffolds are loaded into a 4-mm polyimide sleeve and placed over the ends of the defect. Co-delivery of BMP-2 and lysostaphin results in infection clearance followed by subsequent defect regeneration.

  • Fig. 2 UAMS-1 infection defects treated with BMP-2–loaded lysostaphin-delivering hydrogels significantly improve bone repair.

    Representative μCT reconstructions at 4 and 8 weeks. (A) Quantification of bone volume from μCT imaging at 4 (B) and 8 (C) weeks after implantation. Kruskal-Wallis test with Dunn’s multiple comparisons test. Means ± SD. n = 12 to 18 per group. *P < 0.05, **P < 0.01, ***P < 0.001. (D) Defect bridging was assessed semiquantitatively using the following scale: 0, no bone formation; 1, less than half of the defect; 2, greater than half of the defect; 3, defect bridged. Kruskal-Wallis with Dunn’s multiple comparisons test. n = 12 to 18 per group. *P < 0.05, ***P < 0.001, ****P < 0.0001. (E) Functional healing was assessed using torsion to failure testing. The average torsion to failure values for healthy mouse radii are plotted as horizontal red lines (0.0032 ± 0.0003 N·m), as reported by Shekaran et al. (F) Torsional stiffness was then calculated for all samples. Means ± SD. n = 8 to 11 per group. One-way analysis of variance (ANOVA) with Tukey’s post hoc test for torsion to failure and Kruskal-Wallis with Dunn’s multiple comparisons test for torsional stiffness. (G) Mouse radii were sectioned and stained with H&E and safranin-O/fast green (Saf-O/FG). One sample was randomly selected and prepared per experimental group. Representative images are displayed.

  • Fig. 3 BMP-2–loaded lysostaphin-delivering hydrogels restore the local inflammatory environment to a regenerative state 1 week after implantation.

    Segmental defects were created, hydrogel scaffolds infected with UAMS-1 with or without lysostaphin as well as sterile gels were implanted, and the inflammatory response was assessed using a multiplexed cytokine array assay 1 week later. (A) Hierarchical cluster analysis of cytokine profiles using the Ward method. (B) PCA of the array data. (C to J) Cytokines with statistically different tissue levels as determined using two-way ANOVA with Bonferroni correction for multiple comparisons. Means ± SD. n = 6 to 7 per group. *P < 0.05, ***P < 0.001, ****P < 0.0001. ns, not significant.

  • Fig. 4 Cytokine profile of BMP-2–loaded lysostaphin-delivering hydrogels 4 weeks post-operatively.

    Segmental defects were created, hydrogel scaffolds infected with UAMS-1 with or without lysostaphin as well as sterile gels were implanted, and the inflammatory response was assessed using a multiplexed cytokine array assay 4 weeks later. (A) Hierarchical cluster analysis of cytokine profiles using the Ward method. (B) PCA of the array data. (C to G) Cytokines with statistically different tissue levels as determined using two-way ANOVA with Bonferroni correction for multiple comparisons. Means ± SD. n = 6 to 7 per group. *P < 0.05, ***P < 0.001, ****P < 0.0001.

  • Fig. 5 Total number of inflammatory cells at 1 week after implantation of BMP-2–loaded lysostaphin-delivering hydrogels.

    One week following segmental defect creation and implant placement, mice were euthanized, the implant and surrounding tissue were recovered, and flow cytometry was performed to enumerate the total number of inflammatory cells present. (A) Total cells, (B) CD3+ T cells, (C) CD3+CD4+ helper T cells, (D) CD3+CD8+ cytotoxic T cells, (E) CD19+ B cells, (F) F4/80+ macrophages, (G) CD86+ M1 macrophages, (H) CD206+ M2 macrophages, (I) CD11b+ myeloid cells, (J) Ly6G+ neutrophils, (K) Ly6Clow AM monocytes, and (L) Ly6Chigh IM monocytes were enumerated. Data were log-transformed, and ordinary one-way ANOVA with Tukey’s post hoc test was used. Means ± SD. n = 6 to 7 per group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. IM, inflammatory monocyte; AM, anti-inflammatory monocyte.

  • Fig. 6 Total number of inflammatory cells at 4 weeks after implantation of BMP-2–loaded lysostaphin-delivering hydrogels.

    Four weeks following segmental defect creation and implant placement, mice were euthanized, the implant and surrounding tissue were recovered, and flow cytometry was performed to enumerate the total number of inflammatory cells present. (A) Total cells, (B) CD3+ T cells, (C) CD3+CD4+ helper T cells, (D) CD3+CD8+ cytotoxic T cells, (E) CD19+ B cells, (F) F4/80+ macrophages, (G) CD86+ M1 macrophages, (H) CD206+ M2 macrophages, (I) CD11b+ myeloid cells, (J) Ly6G+ neutrophils, (K) Ly6Clow AM monocytes, and (L) Ly6Chigh IM monocytes were enumerated. Data were log-transformed, and ordinary one-way ANOVA with Tukey’s post hoc test was used. Means ± SD. n = 6 to 7 per group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/5/eaaw1228/DC1

    Supplementary Methods

    Fig. S1. Co-encapsulation of UAMS-1 and lysostaphin in the hydrogel system does not affect bacterial viability.

    Fig. S2. BMP-2–loaded lysostaphin-delivering hydrogels exhibit diffusion-mediated and protease-triggered release.

    Fig. S3. Lysostaphin-delivering hydrogels eliminate infection at 1 week.

    Fig. S4. Lysostaphin-delivering hydrogels eliminate infection at 8 weeks.

    Fig. S5. BMP-2–loaded lysostaphin-delivering hydrogels do not show signs of systemic toxicity.

    Fig. S6. Gating strategy for inflammatory cell profiling analysis.

    Fig. S7. Percent of parent inflammatory cells at 1 week after implantation of BMP-2–loaded lysostaphin-delivering hydrogels.

    Fig. S8. Percent of parent inflammatory cells at 4 weeks after implantation of BMP-2–loaded lysostaphin-delivering hydrogels.

    Table S1. Immune cell profiling antibody characteristics.

  • Supplementary Materials

    This PDF file includes:

    • Supplementary Methods
    • Fig. S1. Co-encapsulation of UAMS-1 and lysostaphin in the hydrogel system does not affect bacterial viability.
    • Fig. S2. BMP-2–loaded lysostaphin-delivering hydrogels exhibit diffusion-mediated and protease-triggered release.
    • Fig. S3. Lysostaphin-delivering hydrogels eliminate infection at 1 week.
    • Fig. S4. Lysostaphin-delivering hydrogels eliminate infection at 8 weeks.
    • Fig. S5. BMP-2–loaded lysostaphin-delivering hydrogels do not show signs of systemic toxicity.
    • Fig. S6. Gating strategy for inflammatory cell profiling analysis.
    • Fig. S7. Percent of parent inflammatory cells at 1 week after implantation of BMP-2–loaded lysostaphin-delivering hydrogels.
    • Fig. S8. Percent of parent inflammatory cells at 4 weeks after implantation of BMP-2–loaded lysostaphin-delivering hydrogels.
    • Table S1. Immune cell profiling antibody characteristics.

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