Research ArticleTissue Engineering

Bioengineered human acellular vessels recellularize and evolve into living blood vessels after human implantation

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Science Translational Medicine  27 Mar 2019:
Vol. 11, Issue 485, eaau6934
DOI: 10.1126/scitranslmed.aau6934
  • Fig. 1 Ultrastructure of the HAV matrix.

    (A) Image of a bioengineered HAV (6-mm inner diameter) depicting sections taken for SEM to examine matrix architecture. (B) Outer and (C) inner surface SEMs. (D) Transverse and (E) longitudinal within-wall cross-sectional SEMs. Lower magnification is shown in insets. Scale bars, 60 μm (B and C) or 150 μm (D and E).

  • Fig. 2 Clinical HAV implantation and hemodialysis access.

    (A) Intraoperative image of HAV implantation in the upper arm of a patient with end-stage renal failure for use as an AV conduit. The HAV was tunneled under the skin (yellow arrows) and connected the axillary vein at the venous anastomosis (inset) to the brachial artery (arterial anastomosis). (B and C) Postoperative Doppler ultrasound measurements of mid-HAV diameter, blood flow volume (FV), and time averaged mean velocity (TAMV) during follow-up within the patient population. Data are shown as means ± SE (n = 28 to 59 HAVs) with no significant differences (P > 0.05) as determined by ANOVA and post hoc multiple comparisons tests. (D) Photograph of cannulation of the subdermal HAV (yellow arrows) for hemodialysis. Cannulation was performed multiple times a week to patients starting at 4 or 8 weeks after implantation. Images courtesy of S. Rocko at Duke University Medical Center (A) and J. Turek at Wroclaw Medical University (D).

  • Fig. 3 Representative routine staining of HAVs before and after implantation.

    (A and B) H&E staining of HAV before implantation shows acellular matrix stained pink without purple nuclei, whereas Masson’s trichrome reveals blue-stained collagen fibers (B). (C to J) Stained samples explanted 16 (C and D), 55 (E and F), 100 (G and H), and 200 (I and J) weeks after implantation. a, neoadventitia; m, medial layer; border delineated by a black dashed line. Integration of the HAV neoadventitia with perivascular adipose (G and H, black arrows) and epithelial (I and J, black arrow) tissue. HAV explant sections were taken from mid-graft (16 and 200 weeks) or venous anastomosis (55 and 100 weeks) regions. Insets: Higher magnification of medial layer area denoted by “*”. Scale bars, 50 μm (insets).

  • Fig. 4 Infiltration and maturation of αSMA+ host cells within the implanted HAV.

    Immunofluorescence staining of explanted HAV sections for αSMA (red) and CNN1 (green), a contractile marker of mature SMCs. Developmental maturation indicated by coexpression of CNN1 and αSMA. HAV sections explanted at 16 (A to C), 55 (D to F), 100 (G to I), and 200 (J to L) weeks after implantation. a, neoadventitia; m, medial layer. The boundary between the neoadventitia and medial layers is delineated by a white dashed line. Nuclei (blue) were counterstained with DAPI.

  • Fig. 5 Angiogenic vascularization and luminal endothelialization of the implanted HAV.

    (A) Immunofluorescence staining for the endothelial progenitor marker CD34 revealed numerous angiogenic CD34+ cells (stained green) forming microvessels within the neoadventitia (a) at 16 weeks after implantation. (B) CD34+ cells coexpressed the ubiquitous endothelial marker CD31 (red), but CD31+ cells in some larger microvessels lacked CD34 expression (A and B, white arrows). HAV sections explanted at 55 (C and D), 100 (E and F), and 200 (G and H) weeks after implantation. White arrows in (G) and (H) highlight microvessels with CD34/CD31+ cells. (I) CD31+ endothelial cells (red) lining both the lumen and microvessels of a mid-HAV explant at 44 weeks after implantation. (J to L) Luminal cells express VE-cadherin (CD144, green) and eNOS (red). a, neoadventitia; m, medial layer; a white dashed line was drawn to delineate neoadventitia and medial layers (A to I). Nuclei (blue) were counterstained with DAPI.

  • Fig. 6 Migration and differentiation of Nestin+progenitor cells within the implanted HAV.

    (A to D) Immunofluorescence staining for the intermediate filament Nestin (green) within HAVs explanted at 16 (A), 55 (B), 100 (C), and 200 (D) weeks after implantation. (E to L) Sixteen-week HAV explant samples immunostained for expression of Nestin (green) and αSMA (red) (E to H) or Nestin (green) and CD31 (I to L). Coexpression indicated by overlay of combined staining (yellow) (H and L). CD31+ cells were found (white arrows) on lumen of 16-week HAV explant (I). a, neoadventitia; m, medial layer; a white dashed line was drawn to delineate neoadventitia and medial layers. Nuclei (blue) were counterstained with DAPI.

  • Fig. 7 Progressive influx of MSC-like host cells within the implanted HAV.

    (A to D) Immunofluorescence staining of HAV explants for αSMA and CD90 at indicated time points after implantation. (E) Quantification of CD90+ cells/mm2. Immunofluorescence staining of 200-week HAV explants for CD90 (green) and (F) CD34 (red), (G) CD45 (red), (H) CD44 (red), and (I) CD73 (red) expression. a, neoadventitia; m, medial layer; a white dashed line was drawn to delineate neoadventitia and medial layers in (A) to (D). Nuclei (blue) were counterstained with DAPI. For (E), eight random images were taken from each explant section, and the percentage of cells (DAPI, blue) that expressed CD90 (green) was calculated per area imaged. Data are shown as means ± SD with statistical differences of *P < 0.05, ***P < 0.0005, and ****P < 0.0001 as determined by ANOVA and post hoc multiple comparisons tests.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/11/485/eaau6934/DC1

    Fig. S1. Representative images of all clinical HAV explants stained with H&E.

    Fig. S2. Representative images of all clinical HAV explants immunostained for expression of CD3 and CD20.

    Fig. S3. Representative images of all clinical HAV explants immunostained for expression of CD206, CD80, and CD68.

    Fig. S4. Representative images of all clinical HAV explants immunostained for expression of αSMA and CNN1.

    Fig. S5. Representative images of all clinical HAV explants immunostained for expression of CD31 and CD34.

    Fig. S6. Representative images of all clinical HAV explants immunostained for expression of αSMA and Nestin.

    Fig. S7. Immunofluorescence staining of αSMA and CD31 in 16-week HAV explant.

    Fig. S8. αSMA+/Nestin+ cells participate in HAV self-healing and repair after cannulation injury.

    Fig. S9. Representative images of all clinical HAV explants immunostained for expression of αSMA and CD90.

    Fig. S10. Additional immunofluorescence characterization of CD90+ cells in 200-week HAV explant.

    Fig. S11. Summary diagram illustrating host cell repopulation and maturation within HAV after implantation.

    Table S1. Summary of all clinical HAV explants from phase 2 clinical trials.

    Table S2. Antibodies used and application conditions.

  • This PDF file includes:

    • Fig. S1. Representative images of all clinical HAV explants stained with H&E.
    • Fig. S2. Representative images of all clinical HAV explants immunostained for expression of CD3 and CD20.
    • Fig. S3. Representative images of all clinical HAV explants immunostained for expression of CD206, CD80, and CD68.
    • Fig. S4. Representative images of all clinical HAV explants immunostained for expression of αSMA and CNN1.
    • Fig. S5. Representative images of all clinical HAV explants immunostained for expression of CD31 and CD34.
    • Fig. S6. Representative images of all clinical HAV explants immunostained for expression of αSMA and Nestin.
    • Fig. S7. Immunofluorescence staining of αSMA and CD31 in 16-week HAV explant.
    • Fig. S8. αSMA+/Nestin+ cells participate in HAV self-healing and repair after cannulation injury.
    • Fig. S9. Representative images of all clinical HAV explants immunostained for expression of αSMA and CD90.
    • Fig. S10. Additional immunofluorescence characterization of CD90+ cells in 200-week HAV explant.
    • Fig. S11. Summary diagram illustrating host cell repopulation and maturation within HAV after implantation.
    • Table S1. Summary of all clinical HAV explants from phase 2 clinical trials.
    • Table S2. Antibodies used and application conditions.

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