Research ArticleBIOMATERIALS

Nanofiber-hydrogel composite–mediated angiogenesis for soft tissue reconstruction

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Science Translational Medicine  01 May 2019:
Vol. 11, Issue 490, eaau6210
DOI: 10.1126/scitranslmed.aau6210
  • Fig. 1 Engineering a nanofiber-hydrogel composite with interfacial bonding between nanofiber surface and hydrogel network.

    (A) Schematic of the synthesis and structure of PCL nanofiber-HA hydrogel composite. (B and C) Scanning electron microscopy images of rat native fat tissue (B) and PCL nanofiber-HA hydrogel composite (C) showing that fibers are embedded into the HA hydrogel network (arrowheads). (D) Image showing a PCL nanofiber-HA hydrogel composite (G′ = 250 Pa, left) and an HA hydrogel [oscillatory shear storage modulus (G′0) = 80 Pa, right] constructed from the same 80-Pa HA hydrogel. (E) Image showing that the composite can be injected through a 30-gauge needle. (F and G) G′ of HA hydrogels and nanofiber-hydrogel composites with different PEGDA cross-linker concentrations of 2 mg/ml (F) and 5 mg/ml (G) and fiber amounts (0, 10, and 20 mg/ml) with or without interfacial bonding. The concentration of HA was maintained at 4 mg/ml (n = 3 to 9). Statistical significance was calculated by one-way analysis of variance (ANOVA) with the Dunnett’s post hoc test. Asterisk (*) indicates the comparison between groups with and without fibers (hydrogel control). Hash key (#) denotes the comparison between groups with and without interfacial bonding. ****P < 0.0001, ####P < 0.0001. Data are presented as means ± SEM.

  • Fig. 2 Enhanced cell migration and vascular-like network formation inside composite.

    (A) hASC migration from 200-μm spheroids embedded in the 80- and 150-Pa HA hydrogels and the 150-Pa composite. (B) Quantitative analysis of cell migration distance inside the hydrogels and the composite (n = 4 to 7). (C) hASCs formed 3D connected networks in the 150-Pa composite. (D) Vascular-like network formation in the 80- and 150-Pa hydrogels and the 150-Pa composite. (E) Quantitative analysis of vascular-like network formed inside the hydrogels and the composite (n = 6). Cell morphology was visualized by staining for actin with Alexa Fluor 568 Phalloidin shown in red. Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) shown in blue. Nanofibers were labeled with F8BT shown in green. Scale bars, 100 μm. Statistical significance was calculated by one-way ANOVA with the Dunnett’s post hoc test. Comparison was performed between groups. ##P < 0.01, ####P < 0.0001. Data are presented as means ± SEM.

  • Fig. 3 Improved angiogenesis inside composite after subcutaneous implantation.

    (A) Host vascular cell infiltration in the injected 80- and 150-Pa HA hydrogels and the 150-Pa composite at PODs 7, 14, and 84. Nanofibers were labeled with F8BT shown in green. Cells were stained with RECA-1 for endothelial cells (shown in red) and with DAPI for nuclei (shown in blue). Scale bars, 100 μm. (B and C) Quantitative analysis of the densities of the blood vessels (B) and the blood vessel network (C) inside the hydrogels and the composite (n = 6). Statistical significance was calculated by two-way ANOVA with the Dunnett’s post hoc test. Hash key (#) denotes the comparison between groups. Asterisk indicates the comparison between time points. # or *P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001. Data are presented as means ± SEM.

  • Fig. 4 Infiltration of macrophages into composite and hydrogels and their phenotypes.

    (A) Macrophages infiltrated into 80 and 150-Pa hydrogels and 150-Pa composite at POD 7. Elongated CD206+ macrophages were found inside the composite. Macrophages were stained with CD206 (shown in red) and CD86 (shown in blue). PCL nanofibers were labeled with F8BT shown in green. Scale bars, 100 μm. (B) Macrophages observed inside the composite and the hydrogels at POD 14. Macrophages were stained with CD206 (red), CD68 (gray), and CD86 (red). Cell nuclei were stained with DAPI in blue. Scale bars, 100 μm. (C to E) Quantitative analysis of infiltrated macrophages inside the composite and the hydrogels by flow cytometry (n = 3). Statistical significance was calculated by one-way ANOVA with the Dunnett’s post hoc test to compare between groups. #P < 0.05, ##P < 0.01. Data are presented as means ± SEM.

  • Fig. 5 Composite-mediated high expression of angiogenesis-related genes.

    (A) Heat map analysis of angiogenesis-related gene expression in the 150-Pa composite and the 150-Pa hydrogel at PODs 3, 7, and 14. (B) Relative quantification (RQ) for fold change of angiogenesis-related gene expression between the 150-Pa composite and the PBS control. (C) Comparison of angiogenesis-related gene expression between the 150-Pa composite and the 150-Pa hydrogel at POD 7 (n = 3). Statistical significance was calculated by Student’s t test. #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001. Data are presented as means ± SEM.

  • Fig. 6 Composite-mediated angiogenesis at the rabbit fat tissue defect site.

    (A) General procedure for creating defects in the inguinal fat pad in a rabbit model (steps 1 to 3) and injection with the composite (step 4). Scale bars, 10 mm. (B) Host blood vessel growth into the 80- and 150-Pa hydrogels and the 150-Pa composite at POD 14. Endothelial cells were stained with CD31 in red. CD68+ was used as a pan macrophage marker; CD206+ labeled pro-regenerative macrophages. Macrophages were stained with CD68 (red) and CD206 (red). Cell nuclei were stained with DAPI in blue. Fibers were F8BT-labeled (green). Scale bars, 100 μm. (C) Host blood vessel growth into the 150-Pa hydrogel and the 150-Pa composite at POD 42. Scale bars, 500 μm. (D) CD68-marked pan macrophages; CD206-labeled pro-regenerative macrophages. Scale bars, 100 μm. (E) Quantitative analysis of the distribution of blood vessels inside the 150-Pa composite and the 150-Pa hydrogel at POD 42 (n = 4). Statistical significance was calculated by two-way ANOVA with the Bonferroni’s post hoc test. #P < 0.05, ##P < 0.01, ####P < 0.0001. Data are presented as means ± SEM.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/490/eaau6210/DC1

    Materials and Methods

    Fig. S1. Controlling carboxylic group density on the PCL nanofiber by changing plasma treatment time.

    Fig. S2. Controlling the length of nanofiber fragments and the reproducibility of composites.

    Fig. S3. The molecular structure of HA hydrogel as a comparison for the composite.

    Fig. S4. Characterization of mechanical properties of the composite.

    Fig. S5. Fluorescence image showing the distribution of nanofibers inside the composite.

    Fig. S6. Fluorescence image showing phase separation of carboxyl-functionalized nanofibers from the hydrogel.

    Fig. S7. Vascular-like network formation in HA hydrogels.

    Fig. S8. hASCs cocultured with human umbilical vein endothelial cells inside the 80-Pa hydrogel.

    Fig. S9. Infiltration of host vascular cells into the 150-Pa composite at POD 7.

    Fig. S10. Blood vessels inside the 150-Pa composite at POD 14.

    Fig. S11. Well-formed blood vessel networks inside the 150-Pa composite at POD 84.

    Fig. S12. Vascular maturation inside the 150-Pa composite at PODs 7, 14, and 84.

    Fig. S13. Vascular network inside the 150-Pa composite at POD 84 shown in the 3D image.

    Fig. S14. Macroscopic images of subcutaneously injected 80- and 150-Pa hydrogels and 150-Pa composite at PODs 7 and 14.

    Fig. S15. Perfusable blood vessels observed in the 150-Pa composite examined by indocyanine green angiography.

    Fig. S16. Macrophages infiltrated 80- and 150-Pa hydrogels and 150-Pa composite at POD 14.

    Fig. S17. Composite-mediated macrophage polarization in vitro.

    Fig. S18. Macrophage morphologies inside the 80- and 150-Pa hydrogels and 150-Pa composite under normal conditions.

    Fig. S19. Angiogenesis-related gene expression inside the 150-Pa composite.

    Fig. S20. Comparison of angiogenic gene expression between the composite and the hydrogel with the same G′ of 150 Pa.

    Fig. S21. Composite-mediated tissue regeneration in a rabbit soft tissue defect model.

    Fig. S22. Processes involved in composite-mediated angiogenesis.

    TableS1. Primary antibodies used in this study.

    Data file S1. Individual subject-level data for quantitative analysis.

    References (4248)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Controlling carboxylic group density on the PCL nanofiber by changing plasma treatment time.
    • Fig. S2. Controlling the length of nanofiber fragments and the reproducibility of composites.
    • Fig. S3. The molecular structure of HA hydrogel as a comparison for the composite.
    • Fig. S4. Characterization of mechanical properties of the composite.
    • Fig. S5. Fluorescence image showing the distribution of nanofibers inside the composite.
    • Fig. S6. Fluorescence image showing phase separation of carboxyl-functionalized nanofibers from the hydrogel.
    • Fig. S7. Vascular-like network formation in HA hydrogels.
    • Fig. S8. hASCs cocultured with human umbilical vein endothelial cells inside the 80-Pa hydrogel.
    • Fig. S9. Infiltration of host vascular cells into the 150-Pa composite at POD 7.
    • Fig. S10. Blood vessels inside the 150-Pa composite at POD 14.
    • Fig. S11. Well-formed blood vessel networks inside the 150-Pa composite at POD 84.
    • Fig. S12. Vascular maturation inside the 150-Pa composite at PODs 7, 14, and 84.
    • Fig. S13. Vascular network inside the 150-Pa composite at POD 84 shown in the 3D image.
    • Fig. S14. Macroscopic images of subcutaneously injected 80- and 150-Pa hydrogels and 150-Pa composite at PODs 7 and 14.
    • Fig. S15. Perfusable blood vessels observed in the 150-Pa composite examined by indocyanine green angiography.
    • Fig. S16. Macrophages infiltrated 80- and 150-Pa hydrogels and 150-Pa composite at POD 14.
    • Fig. S17. Composite-mediated macrophage polarization in vitro.
    • Fig. S18. Macrophage morphologies inside the 80- and 150-Pa hydrogels and 150-Pa composite under normal conditions.
    • Fig. S19. Angiogenesis-related gene expression inside the 150-Pa composite.
    • Fig. S20. Comparison of angiogenic gene expression between the composite and the hydrogel with the same G′ of 150 Pa.
    • Fig. S21. Composite-mediated tissue regeneration in a rabbit soft tissue defect model.
    • Fig. S22. Processes involved in composite-mediated angiogenesis.
    • Table S1. Primary antibodies used in this study.
    • References (4248)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). Individual subject-level data for quantitative analysis.

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