Research ArticleBIOENGINEERING

Vasculogenic hydrogel enhances islet survival, engraftment, and function in leading extrahepatic sites

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Science Advances  02 Jun 2017:
Vol. 3, no. 6, e1700184
DOI: 10.1126/sciadv.1700184
  • Fig. 1 Schematic demonstrating vasculogenic, proteolytically degradable synthetic hydrogel structure, islet delivery strategy, and localized gel remodeling within extrahepatic transplant sites.
  • Fig. 2 Localized VEGF enhances vascularization in extrahepatic transplant sites.

    (A) Recipients of PEG-only or VEGF-presenting hydrogels were lectin-perfused at 2 or 4 weeks, and excised grafts were whole mount–imaged. Scale bars, 200 μm. Vascular characteristics of blood vessel fractional area (B), total branch length (C), junction number (D), and branch number per field of view (FOV) (E). Dashed line and shaded region represent average and SEM for pancreas reference, respectively. Minimum to maximum box-and-whisker plots, n = 5 to 7 per group. † versus SUBQ within the same time point (††P < 0.01 and P < 0.05); × versus pancreas control (×××P < 0.001, ××P < 0.01, and ×P < 0.05); evaluated by Kruskal-Wallis nonparametric tests with Dunn’s multiple comparison.

  • Fig. 3 Leukocyte density varies within extrahepatic transplant sites 4 weeks after hydrogel transplantation.

    (A) Extrahepatic transplant site tissue explanted at week 4 after implantation was stained for CD45 (white) and CD11b (magenta) and was imaged for functional vasculature [lectin (green)] and cell nuclei [DAPI (4′,6-diamidino-2-phenylindole) (blue)]. Scale bars, 50 μm. (B) CD11b and CD45 staining was quantified and normalized to FOV area. n = 4 to 7 subjects per group. *P < 0.05, **P < 0.01, and ***P < 0.001, evaluated by Kruskal-Wallis nonparametric tests with Dunn’s multiple comparison.

  • Fig. 4 Vasculogenic hydrogels promote engraftment and function of single pancreatic donor islet graft.

    Gels containing islets, either with or without VEGF, were delivered to extrahepatic sites. (A) Recipients were monitored daily for nonfasting blood glucose values for calculation of average blood glucose (beyond postoperative day 15) (B) and survival curve of diabetes reversal (C). Graft function was further evaluated by IPGTT on day 35 (D) and by monitoring of recipient body weight (E). (F) Whole-mount imaging of lectin (green)–perfused grafts enabled 3D visualization of engrafted islet vascular network. Error bars represent SEM. Scale bars, 100 μm. n = 5 to 8 per group. † versus SUBQ (†††P < 0.001, ††P < 0.01, and P < 0.05); $ versus SBM within the same group (control or VEGF) ($$P < 0.01). Blood glucose averages were evaluated by one-way analysis of variance (ANOVA), and survival curve analysis was performed using log-rank (Mantel-Cox) test.

  • Fig. 5 In vivo bioluminescent islet tracking allows real-time monitoring of islet survival.

    (A) Gels containing Luc+GFP+/B6 hybrid islet grafts, either with or without VEGF, were delivered to extrahepatic sites as demonstrated in the schematic. (B) Representative in vivo bioluminescence images. (C) Recipients were monitored weekly for bioluminescent signal (left) by intraperitoneal luciferin injection, and cumulative bioluminescent data after day 7 demonstrate significantly enhanced survival in EFP-VEGF group. (D) Lectin perfusion at experimental end point allowed visualization of Luc+GFP+/B6 islet graft [GFP (green)] integration with host vasculature [lectin (magenta)]. High-magnification images of EFP/PEG and EFP/PEG-VEGF islets illustrate integration of vasculature with islet organoid structure. (E) Time-to-peak bioluminescent signal serves as an additional measure of graft vascularization over time. Error bars represent SEM. n = 3 to 4 per group. ****P < 0.0001, ***P < 0.005, and **P < 0.01, evaluated by Kruskal-Wallis nonparametric tests with Dunn’s multiple comparison. P < 0.05 versus SUBQ-PEG, evaluated by one-way ANOVA with repeated measures and Dunnett’s multiple comparisons test. Scale bars, 100 μm.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/6/e1700184/DC1

    fig. S1. Gross morphology of extrahepatic transplant sites during gel casting.

    fig. S2. Correlation between site vascularization fractional area and site leukocyte density.

    fig. S3. Blood glucose traces demonstrating individual recipient graft performance in extrahepatic islet transplant sites.

    fig. S4. Long-term engraftment of marginal islet mass in EFP with PEG-VEGF.

    fig. S5. Comparison of reversal in intraportal control islet transplant site and EFP/PEG-VEGF transplant site syngeneic diabetes reversal.

    fig. S6. Survival curve for SUBQ groups.

    fig. S7. Density of vascularized islets by site as demonstrated by lectin labeling.

    fig. S8. Dose-dependent response of Luc+GFP+ islet signal in B6 recipients in EFP site over a 3-week period.

    fig. S9. In vivo imaging of intraportally infused islets.

    fig. S10. Bioluminescence signal kinetics by time point.

    table S1. Exact P values for select comparisons between groups in vascularization metrics analyses.

    table S2. Exact P values for select comparisons between groups in leukocyte presence analyses.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Gross morphology of extrahepatic transplant sites during gel casting.
    • fig. S2. Correlation between site vascularization fractional area and site leukocyte density.
    • fig. S3. Blood glucose traces demonstrating individual recipient graft performance in extrahepatic islet transplant sites.
    • fig. S4. Long-term engraftment of marginal islet mass in EFP with PEG-VEGF.
    • fig. S5. Comparison of reversal in intraportal control islet transplant site and EFP/PEG-VEGF transplant site syngeneic diabetes reversal.
    • fig. S6. Survival curve for SUBQ groups.
    • fig. S7. Density of vascularized islets by site as demonstrated by lectin labeling.
    • fig. S8. Dose-dependent response of Luc+GFP+ islet signal in B6 recipients in EFP site over a 3-week period.
    • fig. S9. In vivo imaging of intraportally infused islets.
    • fig. S10. Bioluminescence signal kinetics by time point.
    • table S1. Exact P values for select comparisons between groups in vascularization metrics analyses.
    • table S2. Exact P values for select comparisons between groups in leukocyte presence analyses.

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