Spontaneous reversal of stenosis in tissue-engineered vascular grafts

Abstract

We developed a tissue-engineered vascular graft (TEVG) for use in children and present results of a U.S. Food and Drug Administration (FDA)-approved clinical trial evaluating this graft in patients with single-ventricle cardiac anomalies. The TEVG was used as a Fontan conduit to connect the inferior vena cava and pulmonary artery, but a high incidence of graft narrowing manifested within the first 6 months, which was treated successfully with angioplasty. To elucidate mechanisms underlying this early stenosis, we used a data-informed, computational model to perform in silico parametric studies of TEVG development. The simulations predicted early stenosis as observed in our clinical trial but suggested further that such narrowing could reverse spontaneously through an inflammation-driven, mechano-mediated mechanism. We tested this unexpected, model-generated hypothesis by implanting TEVGs in an ovine inferior vena cava interposition graft model, which confirmed the prediction that TEVG stenosis resolved spontaneously and was typically well tolerated. These findings have important implications for our translational research because they suggest that angioplasty may be safely avoided in patients with asymptomatic early stenosis, although there will remain a need for appropriate medical monitoring. The simulations further predicted that the degree of reversible narrowing can be mitigated by altering the scaffold design to attenuate early inflammation and increase mechano-sensing by the synthetic cells, thus suggesting a new paradigm for optimizing next-generation TEVGs. We submit that there is considerable translational advantage to combined computational-experimental studies when designing cutting-edge technologies and their clinical management.

Fig. 1.. Scaffold characterization and assembly of the TEVG.

(A) Gross image of a tubular scaffold. Low- and high-magnification transverse SEM images of the scaffold show the PGA fiber bundles, outlined in white. (B) Low-magnification (left) and high-magnification (right) SEM images of the scaffold showing the porous inner and outer surfaces, the middle layer, and the PGA fibers (when dividing the wall sagittally). (C) Schematic drawing demonstrating the method for assembling the TEVG. Briefly, whole bone marrow is aspirated from the iliac crest and mononuclear cells are enriched via density gradient centrifugation and vacuum-seeded on the tubular scaffold, then incubated for 2 hours before implantation as a vascular graft.

Fig. 2.. Clinical performance of the TEVG.

(A) Schematic drawing of the Fontan anatomy as it would appear during a routine angiogram. Representative angiograms (anteroposterior view) of the TEVG obtained (B) before angioplasty and (C) after angioplasty in a case of critical stenosis. The TEVG is outlined with a dotted white line.

Fig. 3.. Computational modeling predicts spontaneously reversible TEVG stenosis.

(A) Key equation within the overall computational model of TEVG development that accounts for changes in overall graft mass via changes in the rates of production m^α and removal q^α of different constituents α, each of which can depend on the inflammatory burden (superscript i), due to the foreign body response, and/or mechanobiological responses (superscript m), due to deviations Δ in circumferential wall stress t_θ and luminal wall shear stress τ_w from homeostatic values. Here, f,$\widehat{f}$ and g,$\widehat{g}$ denote general functions, given in the Supplementary Materials. (B) Table of four key model parameters and possible ranges identified from previous experiments in immunocompetent and immunocompromised mice to bound the possible inflammation-driven process of neovessel formation. (C to J) Parametric studies for these four key parameters: δ modulates the onset and duration of the inflammatory response, β dictates the shape and skew of the inflammatory time course, $K_{\mathrm{h}}^{\mathrm{i}}$ controls the peak rates of inflammatory neotissue production and degradation, and $K_{\max }^{\mathrm{i}}$ scales inflammatory effects on neotissue degradation. Each plot focuses on the early evolution, up to 52 weeks after implantation, of normalized luminal diameter (top row) and wall thickness (bottom row) for variations in δ, β, $K_{\mathrm{h}}^{\mathrm{i}}$, and $K_{\max }^{\mathrm{i}}$, with arrows showing the direction of increasing values given in the table above.

Fig. 4.. TEVG stenosis spontaneously reverses in an ovine model.

(A) Serial angiographic images of a representative ovine TEVG 1 week, 6 weeks, 6 months, and 1 year after implantation. Anastomoses are identified with white arrowheads, and the TEVG is identified by a white dotted outline. White arrow indicates direction of blood flow. (B) Image reconstructions of serial IVUS and angiographic measurements of luminal area, wall thickness, and length of the TEVG, confirming the development of stenosis at 6 weeks but spontaneous resolution by 6 months after implantation. White arrow indicates direction of blood flow. When based on lamb data, the model simulations (solid lines) for the midgraft (C) luminal diameter and (D) wall thickness fit well the hydraulic diameters calculated from the IVUS measurements (symbols) over the 1-year study. Ovine IVUS measurements are represented as means ± SD (n = 22 at 1 week, n = 22 at 6 weeks, n = 20 at 6 months, n = 15 at 1 year).

Fig. 5.. Stenosis arises from excessive inflammation-driven neotissue formation.

(A) Low-magnification images of hematoxylin and eosin (H&E) staining confirm findings from the in vivo imaging that stenosis results from the formation of neotissue on the inner surface of the scaffold and within the wall, causing wall thickening and luminal narrowing. Green solid lines indicate the neovessel lumen, blue dotted lines indicate luminal neotissue formation, and solid blue lines indicate the abluminal surface of the scaffold. Scale bar, 2.0 mm. (B) Quantitative histological analysis of wall thickness and (C) model simulations of TEVG wall thickness over time (one-way ANOVA with Tukey’s post hoc multiple comparisons test, α = 0.05, *P < 0.05). (D) Staining for αSMA expression. Solid green lines indicate the luminal border of the neovessel. Scale bar, 40 μm. (E) Quantifications of αSMA⁺ area within the vascular neotissue over time and (F) model predictions of smooth muscle cell density (one-way ANOVA with Tukey’s post hoc multiple comparisons test, α = 0.05, *P < 0.05). (G) Staining for CD68⁺ macrophages and (H) quantification (one-way ANOVA with Tukey’s post hoc multiple comparisons test, α = 0.05, *P < 0.05). Remaining scaffold polymer at 6 weeks is outlined in black. (I) Computational model predictions of inflammatory cell density. Plotted data represent means ± SEM (n = 1 at 1 week, n = 2 at 6 weeks, n = 4 at 6 months, n = 2 at 1 year).

Fig. 6.. Extracellular matrix production and remodeling, scaffold degradation, and mechanical properties of the neovessel are consistent with model predictions.

(A) Polarized light images (25×) of PSR-stained transverse sections of the mid-TEVG 1 week, 6 weeks, 6 months, and 1 year after implantation (clockwise). Scale bar, 1.0 mm. White insets highlight 200× polarized light images of the birefringent polymeric scaffold apparent at 1 and 6 weeks, which is subsequently replaced by increasingly thick and aligned collagen fibers at 6 months and 1 year. Scale bar, 40 μm. (B) Quantification of total collagen area fraction from the PSR stain over time compared to the native IVC (one-way ANOVA with Tukey’s multiple comparisons test, α = 0.05, **P < 0.005, ***P < 0.0005, and ****P < 0.0001; plot represents means ± SEM; n = 1 at 1 week, n = 2 at 6 weeks, n = 4 at 6 months, n = 2 at 1 year). (C) Ratio of thick-to-thin collagen fibers in the TEVG over time compared to the native IVC (means ± SEM). n = 1 at 1 week, n = 2 at 6 weeks, n = 4 at 6 months, n = 2 at 1 year. (D) Experimental measurements of the mechanical behavior of the TEVG in response to in vitro pressurization after 1.5 years of development in vivo (symbols represent means ± SD); predicted values from the computational model are shown with a solid line. (E) Predicted graft compliance over 2 to 3 mmHg through 2 years of simulated neovessel development (solid line); the experimentally measured compliance at 18 months is shown by symbol representing mean ± SD.

Fig. 7.. Potential clinical case of reversible TEVG stenosis.

(A) CT images of the TEVG (outer wall outlined with white dots and lumen outlined with yellow dots) from patient 13 from the Japanese trial demonstrating wall thickening and luminal narrowing that presented during the first year after implantation and (B) resolved over the course of several months after treatment with oral anticoagulation (15).