Engineered Tissue-Stent Biocomposites as Tracheal Replacements - PubMed (original) (raw)

. 2016 Sep;22(17-18):1086-97.

doi: 10.1089/ten.TEA.2016.0132.

Sumati Sundaram 2 1, Andrew V Le 1, Angela H Huang 2, Jiasheng Zhang 3, Go Hatachi 1, Arkadi Beloiartsev 1, Michael G Caty 4, Tai Yi 5, Katherine Leiby 2, Ashley Gard 2, Mehmet H Kural 1, Liqiong Gui 1, Kevin A Rocco 1, Amogh Sivarapatna 2, Elizabeth Calle 2, Allison Greaney 2, Luca Urbani 6, Panagiotis Maghsoudlou 6, Alan Burns 6 7, Paolo DeCoppi 6, Laura E Niklason 2 1

Affiliations

Engineered Tissue-Stent Biocomposites as Tracheal Replacements

Liping Zhao et al. Tissue Eng Part A. 2016 Sep.

Abstract

Here we report the creation of a novel tracheal construct in the form of an engineered, acellular tissue-stent biocomposite trachea (TSBT). Allogeneic or xenogeneic smooth muscle cells are cultured on polyglycolic acid polymer-metal stent scaffold leading to the formation of a tissue comprising cells, their deposited collagenous matrix, and the stent material. Thorough decellularization then produces a final acellular tubular construct. Engineered TSBTs were tested as end-to-end tracheal replacements in 11 rats and 3 nonhuman primates. Over a period of 8 weeks, no instances of airway perforation, infection, stent migration, or erosion were observed. Histological analyses reveal that the patent implants remodel adaptively with native host cells, including formation of connective tissue in the tracheal wall and formation of a confluent, columnar epithelium in the graft lumen, although some instances of airway stenosis were observed. Overall, TSBTs resisted collapse and compression that often limit the function of other decellularized tracheal replacements, and additionally do not require any cells from the intended recipient. Such engineered TSBTs represent a model for future efforts in tracheal regeneration.

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Conflict of interest statement

L.E.N. is a founder and shareholder in Humacyte, Inc., which is a regenerative medicine company. Humacyte produces engineered blood vessels from allogeneic SMCs for vascular surgery. L.E.N.'s spouse has equity in Humacyte, and L.E.N. serves on Humacyte's Board of Directors. L.E.N. is an inventor on patents that are licensed to Humacyte and that produce royalties for L.E.N. L.E.N. has received an unrestricted research gift to support research in her laboratory at Yale. Humacyte did not fund this study and did not influence the conduct, description, or interpretation of the findings in this report.

Figures

<b>FIG. 1.</b>

**FIG. 1.

Creation of the engineered TSBT. (A) Clinical-grade metal nitinol stent, 6 mm diameter. (B) Stent–polymer (polyglycolic acid) composite scaffold. (C) Bioreactor culture of smooth muscle cells on stent–polymer scaffold. (D) Engineered cellular tissue–stent composite at the end of 8 weeks. (E) Final product for tracheal replacement—4-mm diameter decellularized engineered TSBT. (F) Gross picture of a 16-mm diameter TSBT, of a size suitable for clinical airway repair. TSBT, tissue–stent biocomposite trachea.

<b>FIG. 2.</b>

**FIG. 2.

Biochemical characterization of engineered TSBTs. (A) Collagen quantified by hydroxyproline assay for native rat, decellularized native rat trachea, and TSBTs. (B) sGAGs quantified by Blyscan assay for native rat trachea, decellularized native rat trachea, and TSBT. ns, not significant; #p < 0.01 (n = 4 for native rat trachea, n = 3 for decellularized native rat trachea, n = 3 for TSBT). sGAGs, sulfated glycosaminoglycans.

<b>FIG. 3.</b>

**FIG. 3.

Angiogenic testing of TSBTs: chorioallantoic membrane assay. (A) Representative images of negative control (nylon membrane), nylon loaded with VEGF 200 ng/mL and engineered matrix scaffold TSBT placed in ovo at the end of 7 days of incubation. (B) Number of vessels attracted toward the matrices at days 0 and 7 postimplantation, significant differences between all groups at day 7. *p < 0.05 (day 0: n = 4 for nylon, n = 4 for nylon + VEGF, n = 3 for TSBT scaffold; day 7: n = 3 for nylon, n = 4 for nylon + VEGF, n = 3 for TSBT scaffold). VEGF, vascular endothelial growth factor.

<b>FIG. 4.</b>

**FIG. 4.

Mechanical characterization of 4-mm diameter TSBTs and rat tracheas: (A, C) Representative stress–strain curves for tensile (A) and compressive (C) loads comparing TSBT (green curve) with native (blue) and decellularized native (red) rat tracheas. Tensile and compressive moduli for TSBT and rat trachea are shown in (B) and (D), respectively. Moduli computed from slope of curves beyond 60% strain. For tensile and compression studies, n = 3 for all samples. # denotes p < 0.05.

<b>FIG. 5.</b>

**FIG. 5.

In vivo testing of TSBT in a rat model. (A) End-to-end implantation of a 4-mm TSBT as a tracheal replacement. (B) Explant at 8 weeks showing metal stents and remodeled tissue with new angiogenic ingrowth. (C) Luminal view of explant at 8 weeks showing a patent graft.

<b>FIG. 6.</b>

**FIG. 6.

Remodeling of TSBT in a rat model over 8 weeks. H&E staining of a preimplant TSBT indicating acellular nature of graft (A, D). Remodeled with cellular infiltration throughout at weeks 2 and 8 postimplant (B, C). Arrowheads in (B, C) indicate luminal surface of TSBTs. Luminal cell repopulation at 2 weeks shows cuboidal cells (E, enlarged solid red box from B) and at 8 weeks shows ciliated cells (F, enlarged solid blue box from C). Repopulation of the TSBT walls shows areas that appear to be vascularized developed at 2 weeks (G, enlarged dotted red box from B), and at 8 weeks (H, enlarged dotted blue box from C). Arrowheads in (G, H) indicate microvessels. Asterisk (*) indicates location of metal stent struts. Scale bars: (A), 500 μm; (B–D), 100 μm; (E, F), 25 μm; (G, H), 100 μm. H&E, hematoxylin and eosin.

<b>FIG. 7.</b>

**FIG. 7.

Immunofluorescence of remodeled TSBT in a rat model at 1 week. Immunofluoresence staining for (A) E-cadherin (red), a pan-epithelial marker and (B) Alpha smooth muscle actin (red), a marker for smooth muscle cells. Scale bar: (A), 100 μm; (B), 50 μm. L, lumen of TSBT.

<b>FIG. 8.</b>

**FIG. 8.

In vivo testing of TSBT in a primate model (A) Remodeling over 7 weeks. Low power H&E staining of TSBT in a nonhuman primate showing cross section of TSBT wall at (B) 2 weeks and (C) 7 weeks. Asterisk (*) indicate stent struts, arrowheads point to luminal surface. H&E of TSBT shows a simple cuboidal epithelium at (D) 2 weeks and multilayer pseudostratified columnar luminal cells at 7 weeks in primate explants (E). Scale bars: (B, C), 100 μm; (D, E), 20 μm.

<b>FIG. 9.</b>

**FIG. 9.

Immunofluorescence of remodeled TSBT in a primate model at 7 weeks. Immunofluoresence staining for (A, C) E-cadherin (red), a pan-epithelial marker and for (B, D) cytokeratin5 (red), a marker for basal-like cells. CK5 staining observed at the base of the luminal cell layer. DAPI-stained nuclei are blue. (C) is enlarged from dotted area in (A); (D) is enlarged from dotted area in (B). Scale bars: (A–D) 75 μm.

<b>FIG. 10.</b>

**FIG. 10.

Microvessels formed in remodeled TSBT at 7 weeks in a primate model. (A) αSMA (red) positive and (B) vWF (red) positive cells lining microvascular structures of the remodeled TSBT. Nuclei in (A, B) are blue. (C) H&E staining of remodeled wall of TSBT from a primate showing vascular structures. Scale bars: (A–C) 75 μm. αSMA, alpha smooth muscle actin; vWF, von Willebrand factor.

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