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© 2020 Wiley-VCH GmbH Supporting Information for Adv. Healthcare Mater., DOI: 10.1002/adhm.202000782 Optimizing Bifurcated Channels within an Anisotropic Scaffold for Engineering Vascularized Oriented Tissues Yongcong Fang, Liliang Ouyang, Ting Zhang,* Chengjin Wang, Bingchuan Lu, and Wei Sun

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© 2020 Wiley-VCH GmbH

Supporting Information for Adv. Healthcare Mater., DOI: 10.1002/adhm.202000782 Optimizing Bifurcated Channels within an Anisotropic Scaffold for Engineering Vascularized Oriented Tissues Yongcong Fang, Liliang Ouyang, Ting Zhang,* Chengjin Wang, Bingchuan Lu, and Wei Sun

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Supporting Information

Optimizing Bifurcated Channels within an Anisotropic Scaffold for Engineering

Vascularized Oriented Tissues

Yongcong Fang, Liliang Ouyang, Ting Zhang*, Chengjin Wang, Bingchuan Lu, Wei Sun

Table of Contents

Supplementary Methods

Oxygen Diffusion Coefficient Measurement

Hydraulic Permeability Measurement

Mechanical Analysis

Immunostaining

DNA Quantification

Supplementary Figures

Figure S1. The schematic diagram of the fabrication of the cellular construct.

Figure S2. Sacrificial template fabrication and channel characterization.

Figure S3. Morphology of porous polymer membrane.

Figure S4. The characterization of lumen endothelialization.

Figure S5. Characterization of C2C12s-seeded oriented scaffolds by staining.

Figure S6. Simulation of shear stress distribution within bifurcated channels.

Supplementary Tables

Table 1. Parameters used to simulate oxygen concentration profiles.

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Supplementary Methods

Oxygen Diffusion Coefficient Measurement

An experimental apparatus modified from a conventional diffusion measurement system

was constructed to determine the effective oxygen diffusion coefficient (De,s). Briefly, the

apparatus consisted of two chambers filled with culture medium and separated by the

porous scaffold. The donor chamber was supplied with fresh saturated culture medium

via a peristaltic tubing system to maintain a saturated oxygen concentration. This setup

allowed oxygen molecules to diffuse from the donor chamber to the receiver chamber

through the porous scaffold. The apparatus was placed on a stirring hot plate to maintain

the temperature at 37 °C during measurement. The change in oxygen concentration in the

receiver chamber was measured using an oxygen probe (RDO™ Optical Dissolved

Oxygen Sensor, Thermo Scientific, USA) every 5 min for 100 min with an initial oxygen

concentration below 2 mg L-1

. Although the variation was less than 0.5 mg L-1

, the

saturated oxygen concentration in the donor chamber was also measured for each

experiment. The measured oxygen concentration data were used to calculate the effective

oxygen diffusion coefficient (De,s).

Hydraulic Permeability Measurement

An experimental setup based on Darcy’s Law was prepared to determine hydraulic

permeability. Briefly, the scaffold was fitted into a chamber connected to the water

reservoir via a silicon tube using a rubber gasket. Water flow from the water reservoir to

the chamber was provided by gravity. The fixed vertical distance from the chamber to the

surface of the water in the reservoir resulted in a constant pressure. The flow rate was

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calculated by measuring the volume of the water that accumulated in the beaker over 30 s

and hydraulic permeability was calculated from at least three scaffolds.

Mechanical Analysis

Scaffold mechanical properties were evaluated using the uniaxial stretch with a

mechanical testing machine (ELF3200, Bose, USA). Scaffold samples were cut into

rectangular pieces (10 mm × 15 mm × 2 mm), hydrated in PBS solution until testing, then

secured at opposite ends onto customized clamps. Samples were preconditioned for ten

cycles at a 0.05 mm min-1

rate with a 0.05 N preload. This was followed by 0.1 mm min-1

uniaxial stretching. The load and displacement data were collected at a frequency of 2 Hz.

The stress-strain plot was processed and the elastic modulus was calculated using 5%–

20% strain.

Immunostaining

Immunostaining was performed to evaluate the morphological features of cellular

constructs. Briefly, the cellular constructs were rinsed with PBS, fixed with 4%

paraformaldehyde solution, rinsed with PBS again, then permeabilized with 0.1% Triton

X-100 followed by blocking with 10% bovine serum albumin (Calbiochem, USA). To

visualize endothelial cells, the HUVEC-laden samples were incubated with anti-CD31

(1:100 dilution, Rabbit mAb, Abcam) primary antibody at 4°C overnight, followed by

PBS washing and further incubation with goat anti-rabbit secondary antibody (1:500

dilution, Alexa Fluor 594, Invitrogen) diluted in 1% BSA for 2 h at room temperature.

Samples were then counterstained with 4′,6-diamidino-2-phenylindole (DAPI, GBI Labs,

0.5 mg mL-1

) for 15 min before imaging. Cellular samples were optionally stained with

Phalloidin-iFluor 488 (0.5 mg mL-1

, Abcam) for 1 h at room temperature to visualize

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cytoskeleton. Confocal microscopy was performed using an inverted Zeiss LSM 880

(Zeiss, Germany) microscope with 4× and 10× magnification objectives.

DNA Quantification

The cellular constructs were harvested on day 3. Total DNA was extracted by digesting

the cellular constructs in a proteinase K solution (Yeasen, China) at 56 °C for 16 h,

followed by three freeze-thaw cycles (10 min at -70 °C and 10 min at 36 °C, respectively).

After centrifugation at 13,000 rpm and 4 °C for 15 min, the supernatant was removed and

the DNA pellets were resuspended in distilled water. Finally, the DNA content was

measured at 260 nm using a spectrophotometer (ND1000, NanoDrop Technologies,

USA).

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Supplementary Figures

Figure S1. The schematic diagram of the fabrication of the cellular construct.

I) Aqueous PVA solution casted on the PDMS mold and allowed to dry overnight; II-III)

The dried PVA layer (II) gently demolded to generate the PVA template (III); IV) The

PVA template further coated with polymer membrane by immersion in a 25 mg mL-1

solution of poly(D, L-lactide-co-glycolide) (PLGA) in chloroform; V) Chitosan/Collagen

solution casted in custom-made mold with PVA template embedded and frozen at -20 ℃

under unidirectional temperature field; The frozen scaffold freeze-dried for 48 h to

generate oriented macro-pores; VI) The scaffold crosslinked with sodium-polyphosphate

(TPP) solution, and dissolved in distilled water to generate channel network; VII)

Endothelial cells seeded into the channel networks by perfusion seeding method; IX)

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After incubation, C2C12s seeded into the porous zone by a combination of needle array-

assisted perfusion seeding and pipette seeding on the surface to obtain a uniform cell

distribution. The bioreactor was perfused with mixed culture medium through the channel

network to provide nutrient and oxygen to the cellular construct.

Figure S2. Sacrificial template fabrication and channel characterization.

The fabricated PVA templates with bifurcation levels A) 2 and B) 3. C) The

chitosan/collagen scaffold after crosslinking and sacrificial template dissolution (scale

bar: 1 mm). D) Changes in diameter between the sacrificial templates and bifurcated

channels.

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Figure S3. Morphology of porous polymer membrane.

The sizes and weight ratios of NaCl porogen particles: A, B) 10–25 μm, 50 wt%, C) 25–

50 μm, 50 wt%, D) 10–25 μm, 70 wt%, and E) 10–25 μm, 90 wt%, respectively. Scale

bars: A) 100 µm, B-E) 25 µm.

Figure S4. The characterization of lumen endothelialization.

A) Live/dead staining on day 0 showing HUVECs distribution. B) F-actin

immunostaining on day 3 showing HUVECs morphology. C,D) CD31 immunostaining

on day 3 revealing the specific tight junction of HUVECs. E,F) SEM images showing

channel surface morphology on day 3 with inset of HUVECs distribution. The white

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arrows pointing to the micro-holes on the channel surface. G) Reconstructed tubular

structure from F-actin immunostaining on day 3. H) F-actin immunostaining on day 7

showing a confluent monolayer lining the channel surface. Scale bars: A-H) 100 µm.

Figure S5. Characterization of C2C12s-seeded oriented scaffolds by staining.

Live/dead staining on day 1 showing cell viability at A) cross-section and B-C)

longitudinal sections. B) and C) indicate positions near and away from the channels,

respectively. D) F-actin immunostaining on day 3 at a cross-section showing the cell

morphology near the channels. Cross-section images of pimonidazole staining on day 3

showing hypoxic cells E) near and F) away from the channels. Scale bars: A) 500 µm, B-

C, E-F) 100 µm, D) 200 µm..

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Figure S6. Simulation of shear stress distribution within bifurcated channels.

Supplementary Tables

Table 1. Parameters used to simulate oxygen concentration profiles.

Parameters Values Sources

Hydraulic Permeability_Parallel: K_P 5.05×10-12

m2 measured

Hydraulic Permeability_Vertical: K_V 1.11×10-12

m2 measured

Effective Diffusion Coefficient_Parallel: D_P 1.52×10-5

cm2 s

-1 measured

Effective Diffusion Coefficient_Vertical: D_V 2.32×10-6

cm2 s

-1 measured

Porosity 0.72 measured

Oxygen Diffusion Coefficient in Culture Medium: D_M 3.83×10-5

cm2 s

-1 ref 28,b

Maximum Oxygen Uptake Rate: V_O2max 2.5×10-17

mol cell-1 s

-1 ref 28,b

Michaelis−Menten Constant: K_m 6.875 μmol L-1 ref 28,b

Oxygen Concentration at the Boundary: C_oxy 222.5 μM ref 28,b

Inlet Oxygen Concentration: C_inlet 222.5 μM ref 28,b

Viscosity of Culture Medium: μ 8.4×10-4 kg m

-1 s

-1 ref 28,b

Cell Seeding Density: N_cell 5×106 cell cm

-3 set

Diameter at the inlet: D_inlet 1.0×10-3 m set

Flow Velocity at the inlet: V_inlet 2.12×10-4 m s

-1 set


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