Research ArticleTissue Engineering

Bioengineered vocal fold mucosa for voice restoration

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Science Translational Medicine  18 Nov 2015:
Vol. 7, Issue 314, pp. 314ra187
DOI: 10.1126/scitranslmed.aab4014
  • Fig. 1. Isolation, purification, and expansion of primary VFF and VFE from human VF mucosa.

    (A) Schematic showing general procedure for fibroblast and epithelial cell isolation and purification from VF mucosa. (B) Morphology of primary VFF and VFE in monolayer culture before first passage [10 or 21 days (d) after seeding] and at passage 3 [P3; hematoxylin and eosin (H&E) staining]. Scale bars, 30 μm. (C) Expression of P4HB, CD90, pan-KRT, KRT14, KRT19, and CD227 in VFF and VFE at P3. Positive/negative gates [versus fluorescence minus one (FMO) control] are shown in gray; low/high gates are shown in black. Data are means ± SEM (n = 4 to 12). P values were calculated using a Student’s t test; n.s., not significant. (D) Representative CD90/CD227 double staining. (E) VFF and VFE population doubling times from P1 to P6. Data are means ± SEM (n = 4). The P value was calculated using analysis of variance (ANOVA).

  • Fig. 2. Assembly of engineered human VF mucosa.

    (A) H&E and Movat pentachrome (connective tissue) staining of engineered and native mucosae. Black arrows indicate basal VFE cytoplasmic projections extending into the lamina propria. Scale bar, 100 μm; 40 μm (insets). (B) Immunofluorescence images showing P4HB, COL4, and CDH1 staining patterns. White arrows indicate P4HB+ VFE, COL4+ basement membrane and luminal epithelial structures, and CDH1+ VFE. White arrowheads indicate P4HB+ VFF and COL4+ VFF and vascular basement membrane structures in the lamina propria. Scale bar, 50 μm. DAPI, 4′,6-diamidino-2-phenylindole. (C) Venn diagram summarizing proteome coverage in engineered mucosa compared to native mucosa and scaffold only. FDR, false discovery rate. (D) Enrichment analysis of the engineered mucosa proteome. Enriched gene ontology terms are depicted as nodes connected by arrows that represent hierarchies and relationships between terms. Node size is proportional to the number of assigned proteins; node color represents the adjusted P value (calculated using BiNGO, n = 3) corresponding to enrichment. Functionally related ontology terms are grouped using colored ovals (green, biological process; red, molecular function; blue, cellular component). Organogenesis/morphogenesis and ECM terms are enlarged for better visualization in fig. S5. (E) Heat maps summarizing NSAF-based quantification of proteins associated with the organogenesis/morphogenesis and ECM ontology terms. A corresponding list of proteins and fold changes is presented in table S4. (F) Rheologic data showing elastic (G′) and viscous (G″) moduli of engineered mucosa compared to native mucosa and scaffold only. Data are means ± SEM (n = 4 to 12). P values (comparison of slopes) were calculated using ANOVA. f, frequency.

  • Fig. 3. Proteomic-based analysis of engineered VF mucosa compared to its isolated subcomponents.

    (A) Venn diagram summarizing proteome coverage across conditions. (B) Volcano plot summarizing NSAF-based protein quantification in engineered mucosa versus VFF in scaffold (red) and VFE on scaffold (blue). The dashed rectangle denotes cutoff criteria for protein overrepresentation in engineered mucosa compared to the other conditions. Adjusted P values were calculated using a Student’s t test (n = 3). (C) Summary of enriched biological process terms associated with the protein set exclusive to engineered mucosa or overrepresented in engineered mucosa compared to both VFF in scaffold and VFE on scaffold. The table lists the three most highly represented terms (adjusted P values were calculated using BiNGO, n = 3; postprocessing was performed using REViGO), as well as the mechanistically relevant epidermis (in the context of mucosa, epithelium) development term. A complete list of enriched terms is presented in table S6. The heat map shows the relative abundance of overrepresented proteins that map to these terms of interest. (D) Immunohistochemical validation of overrepresented proteins LAMA5 (costained with COL4), KRT5, and JUP (costained with CDH1) in engineered and native VF mucosae. White arrows indicate KRT5+ VFE; white arrowheads indicate COL4+ signals in the deep epithelium and JUP+ VFE; and yellow arrows indicate LAMA5+COL4+ basal VFE in engineered mucosa, LAMA5+COL4+ basement membrane structures in native mucosa, and CDH1+JUP+ VFE. Scale bar, 50 μm; 25 μm (inset). (E) Transmucosal electrical resistance. Data are means ± SEM (n = 4). P values were calculated using ANOVA.

  • Fig. 4. Ex vivo physiologic performance of engineered VF mucosa in a canine excised larynx.

    We created human-sized VF mucosae and evaluated their physiologic performance in a large-animal (canine) excised larynx (fig. S7). (A) Aerodynamic data showing Pth, Ps, and U relationships (that is, Rg), as well as 𝒫aero and 𝒫ac relationships (that is, Eg). P values were calculated using ANOVA. (B) HSDI-based glottal area analysis. Gray arrows indicate the beginning, midpoint, and end point of a representative 5.8-ms vibratory cycle. The yellow dashed ellipse indicates maximum glottal area. The yellow dashed line indicates the scanning line used for subsequent kymography. Scale bar, 3 mm. P values were calculated using ANOVA. (C) Representative kymograms from the larynx presented in (B). Red tick marks and dashed lines indicate open and closed phases of a single vibratory cycle. Yellow dashed lines indicate the upper and lower VF margins (UM and LM). Sinusoidal curve fitting (R2 > 0.98) to the UM and LM is shown for the native and engineered conditions. f0, fundamental frequency. (D) Lateral and vertical phase differences for all larynges and conditions. #, contralateral VF mucosa condition used to calculate lateral phase difference; !, VF mucosa condition contralateral to that for which vertical phase difference is calculated. P values were calculated using ANOVA. (E) Representative acoustic data showing time-domain signals (upper), narrowband spectrograms (center), and phase plots (lower). (F) Qualitative acoustic signal typing for all larynges and conditions. P values were calculated using a χ2 test. Data from a parallel experiment evaluating the ex vivo physiologic performance of human oral mucosa are presented in Fig. 5. Data from the same larynx (n = 5) are plotted in the same color (A, B, D, and F).

  • Fig. 5. Ex vivo physiologic performance of human oral mucosa, compared to that of engineered VF mucosa, in a canine excised larynx.

    The ex vivo setup is shown in fig. S7. (A) Phonation threshold pressure (Pth). P values were calculated using a Student’s t test. (B) HSDI-based glottal area analysis. Ps, subglottal pressure. P values were calculated using ANOVA. (C) Representative kymogram from the larynx presented in (B). Red tick marks and dashed lines indicate open and closed phases of a single vibratory cycle. Yellow dashed lines indicate the upper and lower VF margins (UM and LM). Sinusoidal curve fitting (R2 > 0.98) to the UM and LM is also shown. f0, fundamental frequency. (D) Lateral and vertical phase differences for all larynges and conditions. #, contralateral VF mucosa condition used to calculate lateral phase difference; !, VF mucosa condition contralateral to that for which vertical phase difference is calculated. P values were calculated using ANOVA. (E) Representative acoustic data showing a time-domain signal (upper), narrowband spectrogram (lower) and phase plot (right). (F) Qualitative acoustic signal typing. The P value was calculated using a χ2 test. The engineered VF mucosa data set used for statistical comparisons is presented in complete form in Fig. 4. Data from the same larynx (n = 5) are plotted in the same color (A, B, D, and F).

  • Fig. 6. Immunogenicity of engineered VF mucosa.

    (A) Expression of cell surface markers HLA-ABC, HLA-DR, CD80, CD86, PD-L1 (CD274), and PD-L2 (CD273) in VFF and VFE compared to peripheral blood mononuclear cell (PBMC) control. Data are means ± SEM (n = 5). P values were calculated using ANOVA. (B) The schematic illustrating the experimental approach to evaluate in vivo immunogenicity is in fig. S8. Body mass of NSG mice after hPBL injection compared to no-hPBL control mice (n = 10 to 12). Mice were euthanized after a >15% decrease in body mass and clinical signs of xenogeneic GVHD, which occurred 15 to 21 days after hPBL injection. P values (comparison of total percentage change) were calculated using a Student’s t test. (C) hCD45+mCD45 human lymphocytes in the peripheral blood of NSG mice after hPBL injection. Data are means ± SEM (n = 7 to 8). P values were calculated using a Student’s t test. (D) hCD4+ T helper cell and hCD8+ cytotoxic T cell infiltration of the engineered auto- and allografts 15 days after hPBL engraftment. Dashed black contour lines indicate the boundaries between the engineered human grafts (top) and the mouse kidneys (bottom). Scale bar, 500 μm; 70 μm (insets). (E) hFOXP3 expression by infiltrating hCD4+ T cells in the engineered allograft 15 days after hPBL engraftment. White arrows indicate hCD4+hFOXP3 T helper cells; yellow arrows indicate hCD4+hFOXP3+ regulatory T cells; the white arrowhead indicates an hCD4hFOXP3 cell. Scale bars, 5 μm. The bar graph summarizes cell count data from the engineered auto- and allografts and mouse eyelid, a GVHD-positive control tissue. Data are means ± SEM (n = 3 to 6). P values were calculated using ANOVA. (F) H&E-stained sections showing morphology of the engineered auto- and allografts compared to the mouse eyelid 15 days after hPBL engraftment. Scale bars, 50 μm.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/7/314/314ra187/DC1

    Methods

    Fig. S1. Explant culture of primary human VF mucosal cells.

    Fig. S2. VFF distribution and contractile function in type I collagen scaffold.

    Fig. S3. Assembly of engineered VF mucosa.

    Fig. S4. Additional histologic characterization of engineered and native human VF lamina propria.

    Fig. S5. Enlargement of enriched ontology terms within the engineered VF mucosa proteome.

    Fig. S6. Additional quantitative proteomic analysis of engineered VF mucosa compared to its isolated subcomponents.

    Fig. S7. Ex vivo setup used to test physiologic performance of engineered VF mucosa.

    Fig. S8. Experimental approach used to evaluate in vivo immunogenicity.

    Fig. S9. Additional in vivo graft survival and immunogenicity data.

    Table S1. Human donor demographic, surgical, and diagnostic information.

    Table S2. Lists of proteins identified in LC-MS/MS analysis of scaffold only, VFF in scaffold, VFE on scaffold, engineered VF mucosa, and native VF mucosa.

    Table S3. Gene ontology (biological process, cellular component, and molecular function) terms enriched in the engineered VF mucosa proteome.

    Table S4. Lists of ECM and organogenesis/morphogenesis proteins identified in both engineered and native VF mucosae.

    Table S5. Lists of proteins significantly overrepresented in engineered VF mucosa compared to VFE on scaffold, VFE on scaffold compared to engineered VF mucosa, engineered VF mucosa compared to VFF in scaffold, and VFF in scaffold compared to engineered VF mucosa.

    Table S6. Gene ontology biological process terms enriched in the protein set exclusive to engineered VF mucosa or overrepresented in engineered mucosa compared to both VFF in scaffold and VFE on scaffold.

    Table S7. Antibodies and isotype controls used for flow cytometry.

    Table S8. Antibodies used for immunocytochemistry and immunohistochemistry.

    Movie S1. Physiologic vibratory function in a single larynx after unilateral placement of engineered VF mucosa.

    Movie S2. Representative acoustic output from a single larynx.

    References (3640)

  • Supplementary Material for:

    Bioengineered vocal fold mucosa for voice restoration

    Changying Ling, Qiyao Li, Matthew E. Brown, Yo Kishimoto, Yutaka Toya, Erin E. Devine, Kyeong-Ok Choi, Kohei Nishimoto, Ian G. Norman, Tenzin Tsegyal, Jack J. Jiang, William J. Burlingham, Sundaram Gunasekaran, Lloyd M. Smith, Brian L. Frey, Nathan V. Welham*

    *Corresponding author. E-mail: welham{at}surgery.wisc.edu

    Published 18 November 2015, Sci. Transl. Med. 7, 314ra187 (2015)
    DOI: 10.1126/scitranslmed.aab4014

    This PDF file includes:

    • Methods
    • Fig. S1. Explant culture of primary human VF mucosal cells.
    • Fig. S2. VFF distribution and contractile function in type I collagen scaffold.
    • Fig. S3. Assembly of engineered VF mucosa.
    • Fig. S4. Additional histologic characterization of engineered and native human VF lamina propria.
    • Fig. S5. Enlargement of enriched ontology terms within the engineered VF mucosa proteome.
    • Fig. S6. Additional quantitative proteomic analysis of engineered VF mucosa compared to its isolated subcomponents.
    • Fig. S7. Ex vivo setup used to test physiologic performance of engineered VF mucosa.
    • Fig. S8. Experimental approach used to evaluate in vivo immunogenicity.
    • Fig. S9. Additional in vivo graft survival and immunogenicity data.
    • Table S1. Human donor demographic, surgical, and diagnostic information.
    • Table S2. Lists of proteins identified in LC-MS/MS analysis of scaffold only, VFF in scaffold, VFE on scaffold, engineered VF mucosa, and native VF mucosa.
    • Table S3. Gene ontology (biological process, cellular component, and molecular function) terms enriched in the engineered VF mucosa proteome.
    • Table S4. Lists of ECM and organogenesis/morphogenesis proteins identified in both engineered and native VF mucosae.
    • Table S5. Lists of proteins significantly overrepresented in engineered VF mucosa compared to VFE on scaffold, VFE on scaffold compared to engineered VF mucosa, engineered VF mucosa compared to VFF in scaffold, and VFF in scaffold compared to engineered VF mucosa.
    • Table S6. Gene ontology biological process terms enriched in the protein set exclusive to engineered VF mucosa or overrepresented in engineered mucosa compared to both VFF in scaffold and VFE on scaffold.
    • Table S7. Antibodies and isotype controls used for flow cytometry.
    • Table S8. Antibodies used for immunocytochemistry and immunohistochemistry.
    • Legends for movies S1 and S2
    • References (3640)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mp4 format). Physiologic vibratory function in a single larynx after unilateral placement of engineered VF mucosa.
    • Movie S2 (.mp4 format). Representative acoustic output from a single larynx.

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