Biomimetic human small muscular pulmonary arteries

Abstract

Changes in structure and function of small muscular arteries play a major role in the pathophysiology of pulmonary hypertension, a burgeoning public health challenge. Improved anatomically mimetic in vitro models of these microvessels are urgently needed because nonhuman vessels and previous models do not accurately recapitulate the microenvironment and architecture of the human microvascular wall. Here, we describe parallel biofabrication of photopatterned self-rolled biomimetic pulmonary arterial microvessels of tunable size and infrastructure. These microvessels feature anatomically accurate layering and patterning of aligned human smooth muscle cells, extracellular matrix, and endothelial cells and exhibit notable increases in endothelial longevity and nitric oxide production. Computational image processing yielded high-resolution 3D perspectives of cells and proteins. Our studies provide a new paradigm for engineering multicellular tissues with precise 3D spatial positioning of multiple constituents in planar moieties, providing a biomimetic platform for investigation of microvascular pathobiology in human disease.

Fig. 1. Schematics and optical images showing biomimetic hSMPA fabrication and structural similarity to a human small muscular distal acinar pulmonary artery.

(A) Color photomicrograph of an adult hSMPA at the level of the respiratory bronchiole shown in cross section with hematoxylin and eosin staining. Black arrows indicate the EC of the intimal layer (E) and the VSMCs of the muscularis (M). Scale bar, 20 μm. Original magnification, ×200. This native artery exhibits several important structural characteristics including multicellular layering, curvature, and patterning. (B) Schematic illustration of the biomimetic hSMPA featuring patterning of cells and layering of VSMCs (M), laminin, and ECs (E). (C) Schematic illustration of the highly parallel, multistep patterning and assembly process for biomimetic hSMPA. Germanium (Ge) and bilayers of optically transparent silicon oxide and silicon dioxide (SiO/SiO₂) were deposited on silicon wafers using electron-beam evaporation, followed by adhesive protein patterning and cell layering, which were all achieved in 2D. Upon dissolution of the sacrificial germanium layer in cell culture medium, the 2D bilayer films were released and then self-folded into tubes. Additional fabrication details are shown in the schematic in fig. S1, and snapshots of the roll-up process are shown in fig. S2. Fn, fibronectin; Lm, laminin. (D) Confocal microscope images of tubular constructs with tunable 1 and 2 mm length and protein pattern. Luminal surfaces of the tubular constructs were patterned with fluorescently labeled fibronectin (red) or bovine serum albumin (green). The distribution of protein fluorescence intensity is shown in fig. S8A. For cell culture, fibronectin without fluorescence labeling was used. Scale bar, 500 μm. (E) Epifluorescence images of rhodamine-phalloidin–labeled ECs growing on the luminal surfaces of biomimetic microvessels. Scale bar, 500 μm.

Fig. 2. Micromechanics model illustrating similar flexural stiffness in an ultrathin, high-modulus tube as in a thicker, low-modulus tube.

(A) Left: Schematic of a thin long elastic tube compressed by two equal and opposite radial loads. Right: Finite element snapshot showing the deflection of the tube when a force of 13.2 μN is applied to a tube with a thickness of 1.2 μm. The inset shows the front view of the deformed cylindrical shell. The deflection was enlarged 500 times for visualization. (B) 3D natural log plot of flexural stiffness (ψ; unit: N/m), for tubes with a range of Young’s moduli (E; 1 to 200 GPa) and wall thicknesses (t; 0.5 to 2 μm). The inserted plane shows the flexural stiffness calculated on the basis of Eq. 2, using values from hSMPA (t = 30 μm and E = 500 kPa). The plot shows that a tube composed of a thin wall with a high modulus can have the same compliance as a thicker tube with a low modulus. (C) Analytical predictions of the combination of the wall thickness (t) and Young’s modulus (E) for tubular constructs that yield the same flexural stiffness (ψ) as biological hSMPA (t = 30 μm and E = 500 kPa). The plot illustrates that tubes with wall thicknesses below ~500 nm can be composed of stiff wall materials and yet achieve the same flexural stiffness as thick-walled tubes composed of ultrasoft materials such as hydrogels or the cells and extracellular matrix of native blood vessels.

Fig. 3. 3D reconstructions of biomimetic microvessels with ECs illustrating uniformity of cell coverage and intercellular junctional morphology.

Reconstructions of confocal Z-stacks of biomimetic microvessels populated by HPMECs, featuring the following: (A) VE-cadherin at endothelial adherens junctions (antibody labeling, green), (B) F-actin (phalloidin, red), and (C) merged images that include nuclei [4′,6-diamidino-2-phenylindole(DAPI), gray scale]. Insets in (A) and (B) show the cross-sectional views in the corresponding color channel and demonstrate the amount of overlap due to the roll-up process. Scale bars, 100 μm. A green-magenta rendering of this figure is shown in fig. S6.

Fig. 4. HPMEC and HPASMC are layered in biomimetic hSMPA.

(A) Reconstructions of confocal Z-stacks of biomimetic hSMPA populated by layered cocultures of HPMEC (luminal) and HPASMC. (i) VE-cadherin (antibody labeling, green), (ii) smooth muscle α-actin (α-SMA) (antibody labeling, red), and (iii) merged image including nuclei from both cell types (DAPI, gray scale). Scale bars, 100 μm. (B) (i) 3D view of two-channel confocal imaging of a small region of a biomimetic hSMPA. HPMEC are visualized using anti–VE-cadherin antibody (green), while smooth muscle α-actin antibody labeling (red) shows the HPASMC. (ii) XZ projection demonstrates segregation and layering of these two cellular components in this biomimetic hSMPA. The bottom panel in (ii) is sampled from (i) and exhibits intensity distribution. Scale bars, 20 μm. (iii) Normalized fluorescence intensity is plotted versus relative radial distance from the tube’s lumen. The distance between the two cell layers is approximately 3.5 μm. A green-magenta rendering of this figure is shown in fig. S7.

Fig. 5. Patterned fibronectin on tubular constructs oriented HPASMC adhesion.

(A) Confocal image (side view) of HPASMCs on an unpatterned tubular construct, stained for F-actin (phalloidin, red), smooth muscle α-actin (antibody labeling, green), and nuclei (DAPI, gray scale). The polar plot is based on image analysis of the gray scale F-actin image and shows that without patterning, HPASMCs attached and spread with random orientation. (B) Confocal image (side view) of HPASMCs grown on a patterned tubular construct, stained for F-actin (phalloidin, red), smooth muscle α-actin (antibody labeling, green), and nuclei (DAPI, gray scale). On the basis of image analysis of the gray scale F-actin image, the polar plot shows that F-actin filaments demonstrated alignment in parallel helical structures on the fibronectin-patterned scaffold. Binning in the polar plots is 10°. (C) 3D views of biomimetic microvessels demonstrating tunable variations in orientation angles and patterning periodicity, with labeled F-actin (phalloidin, red), smooth muscle α-actin (antibody labeling, green), and nuclei (DAPI, gray scale). Scale bars in (A to C), 100 μm.

Fig. 6. Cellular longevity and cell signaling demonstrated markedly improved functionality in biomimetic hSMPAs.

(A) Cell viability was calculated as the percentage of live cells compared with baseline (day 3) using the CyQUANT assay. Coculture of HPMEC and HPASMC populations were assayed. Data from flat (red) and tubular constructs (green) were compared. Data are displayed as means ± SEM. (B) HPMECs in the biomimetic microvessels exhibited a fourfold rise in nitric oxide production over the levels observed in HPMEC monolayers in flat culture. Nitrite levels in cell culture medium were determined by using a Sievers bioluminescence nitric oxide analyzer. Nitrite levels were normalized to total protein content for each sample. The numbers in the y axis indicate picomoles of nitrite per micrograms of protein. Medium was collected 48 hours after confluency and medium replacement. Data are displayed as means ± SEM (**P < 0.01). (C) Phosphorylation of eNOS (at Ser¹¹⁷⁷) was found to be greater in HPMECs that were seeded and cultured on biomimetic microvessels than that in cells on flat SiO/SiO₂ films. HPMECs were grown at each condition for 48 hours. Western blots of cell lysates from each population were analyzed with antibodies against phosphorylated eNOS (p-eNOS), total eNOS, and β-tubulin (protein loading control). Data are displayed as means ± SEM (**P < 0.01).