Double network polyurethane-gelatin hydrogel with tunable modulus

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Biological and Medical Applications of Materials and Interfaces

Double network polyurethane-gelatin hydrogel with tunable modulus for high resolution 3D bioprinting Cheng-Tien Hsieh, and Shan-hui Hsu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b10784 • Publication Date (Web): 13 Aug 2019 Downloaded from pubs.acs.org on August 14, 2019

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ACS Applied Materials & Interfaces

Double network polyurethane-gelatin hydrogel with tunable modulus for high resolution 3D bioprinting Cheng-Tien Hsieh1 and Shan-hui Hsu1, 2, *

1Institute

of Polymer Science and Engineering, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei 10617, Taiwan, R.O.C. *Corresponding Author: Shan-hui Hsu; E-mail: [email protected] 2Institute

of Cellular and System Medicine, National Health Research Institutes, Zhunan 35053, Taiwan, R.O.C.

Keywords: bioink, 3DP bioprinting, biodegradable, polyurethane, gelatin

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Abstract Three-dimensional (3D) bioprinting is a technology to print materials (bioink) with cells into customized tissues for regeneration or organoids for drug screening applications. Herein, a series of biodegradable polyurethane (PU)-gelatin hydrogel with tunable mechanical properties and degradation rates were developed as the bioink. The PU-gelatin hydrogel demonstrated good printability in 2431 C and could print complicated structure such as the nose-shape construct. Due to the excellent shear thinning and fast strain recovery properties, the PU-gelatin hydrogel also had long working windows for bioprinting (over 24 h), stacking ability (up to eighty layers), as well as feasibility for high-resolution printing (through an 80 m nozzle). The structure stability of PU-gelatin hydrogel was maintained by two-stage double network formation through Ca2+ chelation and thermal gelation at 37 C without any toxic crosslinking reagent. The compressive modulus of printed PU-gelatin hydrogel constructs was increased in about three-fold by the treatment of CaCl2 solution for 15 min, and was enhanced further after incubation because of the thermal sensitivity of PU at 37 C. Mesenchymal stem cells (MSCs) printed with the PU-gelatin hydrogel through the 80 m nozzle showed good viability, high mobility, and ~200% proliferation ratio (or ~300% proliferation ratio through a 200 m nozzle) in 10 days. Furthermore, the MSC-laden PUgelatin constructs containing small molecular drug Y27632 underwent chondrogenesis in 10 days. The novel series of PU-gelatin hydrogel with tunable modulus, long working window, convenient bioprinting process, and high-resolution printing possibilities may serve as new bioink for 3D bioprinting of various tissues.


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1. Introduction

2

Three-dimensional (3D) bioprinting is an emerging technology that combines

3

viable cells with 3D printing technology to produce a variety of tissue engineered

4

constructs with predefined shape and structures to repair or reconstruct the human

5

tissues and organs, or to produce organoids for drug screening applications.1-3 The

6

development of bioink is one of the most challenging issues in the 3D bioprinting

7

process.4 Bioink requires sufficient strength and stiffness to maintain structural

8

integrity of the ink after printing

9

and normal function of the embedded cells. Conventional materials for 3D bioprinting

10

are aqueous polymers dissolved in water to possess flow and printable behavior6 and

11

are later solidified by photocrosslinking,7-13 or by a change in temperature,8,14 ionic

12

concentration,3,15 or pH values.16 The photocrosslinkable hydrogels usually involve

13

the use of toxic initiators to induce the generation of free radical for crosslinking

14

reaction by UV or visible light exposure, while such UV light, toxic initiators, or free

15

radical may damage the cells.7,11,17 Thermoresponsive hydrogels with good

16

biocompatibility may cause less damage to the embedded cells, but they are often not

17

printed alone because the lack of shear thinning properties18-21, unsuitable gelation

18

temperature, or slow gelation rate that limit the integrity and strength of the stacked

19

patterns.14,22 Rapid gelation was observed in some thermoreversible hydrogels where

1,4,5

as well as to support the survival, proliferation,



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the gelation rate was controlled by the hydrogen bonding interactions between the OH

2

group and the NH group.23 These thermoreversible hydrogels comprised of

3

hydrophobic and hydrophilic polymer chains, and when the temperature increased (75

4

C), the hydrophilic interaction decreased and the hydrophobic interaction increased,

5

causing gel formation. However, the temperature (75 C) was too high for cell

6

printing. Ion-sensitive hydrogels are also found in literature with most studies

7

focusing on sodium alginate printed in an ionic (e.g. calcium) solution to solidify into

8

gel. The printed hydrogel has a high water content, good transparency, and adequate

9

cell survival. However, their mechanical properties are weak and the degradation rate

10

are too fast.3 pH-sensitive hydrogels are mostly based on collagen, which is easy to

11

print but mechanically poor.16

12

Materials used in bioink are either synthetic or natural according to their origins.

13

Synthetic materials include poly(ethylene glycol) (PEG),24,25 poly(caprolactone)

14

(PCL),14,22,26

15

Pluronic®.12,28 Natural materials for bioink include collagen,16,29 fibrin,29 sodium

16

alginate,3,30 hyluronic acid,30 and gelatin.7,11,28 Synthetic materials have the

17

advantages of abundancy, stability, better mechanical properties, and tunable

18

degradation rates, yet have lower cell compatibility. Natural materials, which are

19

usually derived from extracellular matrix, have neutral or positive effects on cells, but

polylactide,14,22,26

poly(ethylene

glycol)


dimethacrylates,27

and

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the mechanical properties are generally poor.31 Bioink should possess adequate

2

biological properties (bioactivity and cytocompatilibilty) as well as proper material

3

properties

4

biodegradation).32-34 The current challenges are mainly the difficulties in

5

simultaneously meeting all these requirements.31,33,35

at

37 C

(printability,

pattern

fidelity,

stable

structure,

and

6

Biodegradable polyurethanes (PUs) have been widely used in the field of

7

biomedicine because of biocompatibility, mechanical properties, and elasticity.36

8

Waterborne biodegradable PUs can have a range of mechanical and degradation

9

properties and may even undergo gelation by varying the composition of soft

10

segments in polymer chain.37,38 Waterborne biodegradable PU nanoparticles (NPs)

11

were recently found to shift macrophage population to M2 phenotype (tissue

12

repair/remodeling),39 a unique feature previously reported for collagen.40 Gelatin is a

13

low-charge-density peptide polymer, which is a denatured product of collagen.

14

Gelatin can form gel with 3D helix structure at temperatures lower than the upper

15

critical solution temperature (UCST),41 and the gelling process is fast, which makes it

16

printable at room temperature but melt at 37 C.15

17

In this study, we combine water-based biodegradable PU NP dispersion and

18

gelatin solution to prepare a PU-gelatin bioink. The PU-gelatin bioink can be loaded

19

with cells to print 3D constructs by a 3D bioprinter. PU has many advantages that 5 ACS Paragon Plus Environment


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include good biocompatibilty, tunable degradation rate and a range of mechanical

2

properties. PU also has good shear thinning properties, elasticity, divalent cation

3

senstivity, and can reduce immune response

4

PU-gelatin bioink. Gelatin is a biopolymer with UCST. When the temperature of the

5

solution is below the UCST temperature, gelatin is in a gel state. The PU-gelatin

6

bioink can be temporarily fixed when the temperature of the bioink is below the

7

UCST of gelatin. The PU-gelatin printed constructs can be fixed further through ion

8

chelation by immersion in a nontoxic divaent ionic soltuion. Formation of double

9

network is later achieved by thermal curing of PU. The PU-gelatin bioink has the

10

advantages of having both thermal and ionic sensitivities without the associated

11

shortcomings.

39

PU may provide good printability for

12

In this work, we demonstrate that the PU-gelatin bioink has excellent rheologcial

13

properties for printing, stacking ability (for over eighty layers), a wide working

14

window, as well as high resolution (~100 m) printing possibility. The PU-gelatin

15

double network hydrogel have good elasticity and mechanical strength (hand

16

holdable), and can support the mesenchymal stem cell (MSC) proliferation. The novel

17

PU-gelatin bioink system also possesses tunable mechanical properties and

18

degradation rates that can meet the requirement of different tissues/organs and be

19

mass-produced for drug screening applications and regenerative medicine. 6 ACS Paragon Plus Environment

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2. Materials and Methods

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2.1. Synthesis of the water-based biodegradable PU dispersion

4

Dispersion of PU NPs with different chemical formulae (PU1PU3) were synthesized

5

by waterborne processes.22,38 All PUs contained about ~67wt% soft segment

6

(polyesters) in the molecular chain. The soft segment of PU1 was contributed by two

7

oligodiols poly(-caprolactone) diol (PCL, Mn = 2000 g mol−1; Sigma, USA) and

8

poly(D,L-lactide) diol (PDLLA, Mn = 1500 g mol−1) in 4:1 molar ratio. This formula

9

was recently observed to undergo thermal gelation at 37 C in 25% aqueous

10

dispersion.22 For PU2, the composition of soft segment comprised only PCL (Mn =

11

2000 g mol−1). For PU3, the composition of soft segment included PCL (Mn = 2000 g

12

mol−1) and polyethylene butylene adipate diol (PEBA diol, Mn = 2000 g mol−1; Greco,

13

Taiwan) in 2:3 molar ratio. In contrast to PU1, the water dispersion of PU2 or PU3

14

did not undergo thermal gelation at 37 C. The three PUs had distinct degradation

15

rates.38 The basic characteristics of these different PUs are summarized in Table S1.

16

The degradation time from the shortest to longest is ranked as PU1 > PU3 > PU2. The

17

Young’s modulus from the lowest to highest is in the order of PU2 > PU1 > PU3.

18

Therefore, we can select different PUs to prepare the PU-gelatin bioink and to

19

fabricate constructs that fit different tissues and organs. To prepare the waterborne PU, 7 ACS Paragon Plus Environment


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soft segment was reacted with hard segment isophorone diisocyanate (IPDI; Evonik

2

Degussa GmbH, USA), 2,2-bis(hydroxymethyl)propionic acid (DMPA; Sigma,USA),

3

and

4

oligodiols/IPDI/DMPA/EDA was 1:3.52:1:1.52. The oligodiol or oligodiols was

5

placed in a four-necked vessel and vigorously stirred (180 rpm) under a nitrogen

6

environment for 30 min at 95 C, which were then mixed with IPDI and tin(II) 2-

7

ethylhexanoate (T-9, Alfa Aesar) catalyst for 3 h. After that, DMPA and methyl ethyl

8

ketone (MEK; J.T. Baker, USA) were subsequently under reflux at 75 C for 1 h and

9

the temperature was cooled down to 50 C. Triethylamine (TEA; R.D.H., USA) was

10

added for neutralization, and the mixture was dispersed in deionized water and

11

stirring at 1100 rpm before EDA was finally added. The residual solvent was

12

completely removed by vacuum distillation. The solid content of the as-obtained PU

13

dispersion was about 30 wt%. As mentioned, PU1 had thermo-responsive properties

14

and formed PU hydrogel at 37 C, but the mechanical properties were not enough for

15

stacking more than 50 layers.14,22 The hydrodynamic diameter (Dh) and zeta potential

16

of various PU dispersions in water were measured by dynamic light scattering (DLS;

17

DelsaTM Nano Submicron particle analyzer, Beck-man Coulter, USA). The molecular

18

weight of PUs was measured by a JASCO gel permeation chromatography (GPC)

ethylenediamine

(EDA;

Tedia,

USA).

The


stoichiometric

ratio

of

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system equipped with a refractive index detector (RI-930) using dimethylacetamide as

2

the eluting solvent.

3 4

2.2. Preparation of the 3D printing ink

5

The novel 3D printing ink was made from a hybrid of PU dispersion and gelatin

6

solution. 12.5% (w/v) PU dispersion and 12.5% (w/v) gelatin (300 bloom, Sigma,

7

USA) were mixed in 80/20 ratio. The cell culture medium [Dulbecco’s modified

8

eagle’s medium-low glucose (DMEM-LG, Gibco, USA) and sodium bicarbonate

9

(Sigma, USA)] were mixed in the PU-gelatin hybrid solution. Finally, the ionic

10

concentration of PU-gelatin hybrid solution was the same as that of cell culture

11

medium.

12 13

2.3. Effects of ions on the gelation behavior of PU-gelatin

14

For test the effect of ionic solution on the curing of gelatin solution, PU dispersion,

15

and PU-gelatin mixture, the ionic solution of 0.2 N KCl, 0.2 N NaCl, 0.2 N CaCl2 and

16

0.2 N BaCl2 were prepared. Each sample was soaked respectively in the ionic solution

17

for curing, and then the treated sample was placed in a 37 C incubator for 2 h for

18

examination of the effect of temperature on the cured gel. The chelation effect of

19

divalent metal ion on PU-gelatin hydrogels was confirmed by the attenuated total 9 ACS Paragon Plus Environment


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reflectance-fourier transform infrared (ATR-FTIR) spectroscopy (Nexus 670,

2

Thermo). The ATR-FTIR samples of PU-gelatin hydrogels before and after the

3

treatment of 0.2N CaCl2 solution for 15 min were dried in an 60 C oven prior to the

4

measurement.

5 6

2.4. Degradation and swelling behaviors of PU-gelatin hydrogels

7

The PU-gelatin constructs were immersed in CaCl2 solution of different concentration

8

(0.05, 0.1, 0.2, 0.4, and 0.8 N) CaCl2 solution for 15 min, rinsed with distilled water,

9

and freeze-dried for 24 h and weighed (Wi). In vitro degradation of these dried PU-

10

gelatin constructs were performed in 37 C phosphate buffer saline (PBS) solution for

11

1, 3, 7, 14, and 28 days. After the specified degradation time, the constructs were

12

retrieved, washed with distilled water, freeze-dried for 24 h, and weighed (Wf). The

13

remaining weight of PU-gelatin constructs (%) was calculated by the formula:

14

remaining weight (%) = Wf/Wi × 100%. In the swelling experiment of PU-gelatin

15

constructs, the dried PU-gelatin constructs were immersed in 37 C PBS solution and

16

incubated for 1, 3, 6, 12, 24, 120, 168 h. At various time intervals, the excessive

17

solution on the surface of PU-gelatin constructs were removed and weighed (Ws). The

18

swelling ratio (%) was calculated by the equation: swelling ratio (%) = (Ws-Wi)/Wi ×

19

100%. 10 ACS Paragon Plus Environment

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2.5. Rheological measurements of PU-gelatin ink

3

The rheological properties of the gelatin solution, PU1 dispersion, and PU1-gelatin

4

mixture were evaluated by a rheometer (HR2, TA Instruments) with a cone geometry.

5

The diameter of the cone was 40 mm and the angle was 2. The temperature sweep

6

test of PU-gelatin bioink were carried out from 1531 C at a temperature sweeping

7

rate of 0.2 C per 1 min, under 1% strain and 1 Hz. The measurement followed

8

dynamic and static modes. In the dynamic mode, the gel properties were measured

9

temperature with the 1% strain and 1 Hz frequency immediately after sample loading

10

to the 37 C plate. Moreover, the strain sweep was carried out at 1 Hz frequency and

11

equilibrated at 25 C for 3 min in a strain range of 02500%. In the static mode, the

12

gel was equilibrated at 25 C for 3 min and the steady shear viscosity was measured

13

in a shear rate range of 03000 s-1. The creep-recovery test protocol followed the

14

literature42. The creep and creep recovery (60 s each) of PU1-gelatin were measured

15

at 25 C in the yield shear stress of ~100 Pa and ~150 Pa.

16 17

2.6. Mechanical properties of hydrogels

18

The dynamic compression moduli of PU-gelatin hydrogels were measured by a

19

dynamic mechanical analyzer (DMA) (Q-800, TA Instruments) with 1% oscillatory 11 ACS Paragon Plus Environment


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strain and 1 Hz of frequency at 25 or 37C. The static compression properties of the

2

PU-gelatin hydrogels were measured by DMA with a static rate (3% strain min−1 ) at

3

25 or 37 C. The tensile properties of PU-gelatin hydrogels were measured by DMA

4

with a static rate (0.2 N min−1 ) at 37 C.

5 6

2.7. Cell culture, cell viability, and bioprinting

7

Human induced pluripotent stem cell (hiPS)-derived mesenchymal stem cells (MSCs)

8

were obtained from human umbilical vein endothelial cells (Bioresource Collection

9

and Research Center, Hsinchu, Taiwan) transfected with Oct 4 and Sox 2 by lentivirus,

10

and then the iPS cells were differentiated to MSCs by replacing the iPS medium with

11

the MSC medium.43,44 The hiPS derived MSCs were cultured in DMEM-LG medium

12

containing 3.7 g L−1 sodium bicarbonate (NaHCO3; Sigma, USA), 1% penicillin-

13

streptomycin (Gibco, USA), 1% L-glutamine (Gibco, USA), and 10% fetal bovine

14

serum (FBS; Caisson Laboratories, USA). Cells were maintained in a 5% CO2

15

incubator at 37 C and the culture medium was refreshed twice a week.

16

Human MSCs with a cell density of 3.6×104 cells per l were added in culture

17

medium, PU1 dispersion, and PU1-gelatin hydrogels. The cell viability of culture

18

medium, PU1 dispersion, PU1-gelatin hydrogel, and PU1-gelatin hydrogels (with

19

pretreatment of 0.2 N CaCl2 for different time (5, 10 15, 20, and 25 min)) were 12 ACS Paragon Plus Environment

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evaluated by the Nucleocounter® NC-3000™ with cell fluorescence analysis. The

2

human MSCs were stained by VitaBright-48™ (VB-48), acridine orange (AO), and

3

propidium iodide (PI). AO and PI stain for total cells and dead cells, respectively. The

4

VB-48 dye (blue) detects the glutathione (GSH) level, which is an early hallmark of

5

cell apoptosis, and thus the VB-48 intensity represents the healthy state of cells. To

6

examine the cell viability for a long time (10 days), MSCs with a cell density of 6×106

7

cells per ml were dyed with PKH26 and added in the PU1-gelatin hydrogel and

8

treated with 0.2 N CaCl2 solution at 15 min. After that the cell-laden hydrogels were

9

incubated with the culture medium and the images were recorded using a real-time

10

Cultured Cell Monitoring System (Astec, CCM-Multi, Japan) for 10 days at 1 h

11

intervals. MSCs with a cell density of 6×106 cells per ml were mixed in PU-gelatin

12

mixture (in cell culture medium). The MSC-containing PU-gelatin mixture was

13

loaded into the syringe for printing. After printing, PU1-gelatin gel treated with 0.2 N

14

CaCl2 solution at 15 min. The parameters including nozzle size (80 m needle-like,

15

200 m cone-like or 320 m micropipette tip (as the control)) (as shown in Figure S1),

16

nozzle temperature (2431 C), air pressure (50300 kPa), and platform temperature

17

(2025 C) were adjusted for successful 3D printing. After printing, cell-laden

18

constructs were soaked immediately in 0.2 N CaCl2 solution for 15 min and moved



1

into the 37 C incubator. The cell-laden constructs were assessed for cell proliferation

2

on 1, 2, 3, 5, 7, and 10 days by the WST-8 assay.

3 4

2.8. Differentiation of MSCs in PU-gelatin 3D printed constructs

5

Cell-laden PU1-gelatin-Y27632 constructs were fabricated for chondrogenesis

6

experiment45,46. Y27632 (25 ppm) was added to the PU1-gelatin ink before printing.

7

The cell-laden PU1-gelatin-Y27632 was cultured in a basal medium for 3 days. After

8

3 days, the basal medium was switched to basal medium with 10 M Y27632 for

9

another 7 days. Meanwhile, the printed cell-laden PU1-gelatin constructs were

10

cultured in the basal medium (as the control group) or chondrogenic induction

11

medium (as the positive group) for 10 days. The chondrogenic induction medium was

12

the basal medium supplemented with 10 ng mL−1 TGF-3 (CytoLab/Peprotech,

13

Rehovot, Israel), 0.1 M dexamethasone (Sigma, USA), 40 g mL−1 L-proline

14

(Sigma, USA), 50 g/mL ascorbate-2-phosphate (Sigma, USA), and 1% insulin-

15

transferrin-selenium (ITS)-premix 100× (Sigma, USA). All of the cell-laden

16

constructs were cultured for 10 days and harvested by Trizol reagent (Invitrogen,

17

USA). The gene expression of aggrecan (Agg), SRY-box containing gene 9 (Sox 9),

18

collagen type I (Col I), collagen type II (Col II), and collagen type X (Col X) were

19

analyzed by StepOnePlus thermo cycler (Applied Biosystems) and DyNAmo Flash 14 ACS Paragon Plus Environment

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1

SYBR Green qPCR Kit (Finnzymes Oy, Espoo, Finland). The expression level of

2

each gene was normalized to that of glyceraldehyde 3-phosphate dehydrogenase

3

(GAPDH).

4 5

2.9. Statistical analysis

6

All of the experimental data were presented as the mean  standard deviation (S.D.).

7

Each kind of experiment with multiple samples (n = 3) was repeated independently to

8

verify the reproducibility. Statistical differences among the experimental groups were

9

determined by analysis of variance followed by one-way ANOVA test. Groups with p

10

values of less than 0.05 were considered to have statistically significant difference and

11

are denoted with asterisks.

12 13

3. Results

14

3.1. Effect of ions on PU-gelatin hydrogels

15

The effects of different ions on the appearance of PU1 dispersion are shown in

16

Figure 1(a). In the experiment, PU1 dispersion was treated by PBS or 0.2 N ionic

17

solution (NaCl, KCl, CaCl2 or BaCl2) for 5 min at 25 C. In the group treated with

18

PBS, NaCl or KCl, the appearance did not change significantly, while in the group

19

treated with CaCl2 or BaCl2, partial gelation was observed. When being moved to a 37 15 ACS Paragon Plus Environment


1

C incubator, the group treated with PBS, NaCl, or KCl remained sol-like, and the

2

group treated with CaCl2 or BaCl2 remained gel-like. The effects of different ionic

3

solution (CaCl2 and BaCl2) on the appearance of gelatin are shown in Figure 1(b).

4

Gelatin was gel-like at 25 C and sol-like at 37 °C. Even after treatment (CaCl2 or

5

BaCl2), gelatin returned to sol-like at 37 °C. Various PUs (PU1PU3) exhibited

6

similar gelation phenomena after CaCl2 treatment Figure S2. All PU-gelatin mixture

7

showed a gel state at 25 °C and a liquid state at 37 °C before CaCl2 treatment, while

8

remained gel at 37 C after CaCl2 treatment Figure 1(c). Figure 1(d) presents the

9

ATR-FTIR spectra of PU1-gelatin hydrogels before and after the treatment of CaCl2

10

solution. The untreated PU1-gelatin hydrogel showed absorption peaks at around

11

1536 (-CONH) and 3302 cm−1 (hydrogen bonding of NH). After the treatment in

12

CaCl2 solution for 15 min, these peaks were shifted to higher wavenumbers (1557 and

13

3341 cm−1). Raman spectra of PU-gelatin bioink before and after chelation by 0.2 N

14

CaCl2 solution are shown Figure S3. The amide I band was observed at 1667 cm-1 in

15

the PU-gelatin bioink before the chelation of Ca2+. After the PU-gelatin bioink was

16

chelated with Ca2+, the band at 1667 cm-1 became less intense.

17 18

3.2. Rheological properties of PU-gelatin hydrogels


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1

The rheological properties of gelatin are shown in Figure S4 and S5. The complex

2

viscosity of gelatin solution was higher than that of PU dispersion at 25 °C. The PU

3

dispersion had a very low complex viscosity before gelation. The rheological

4

properties of the PU1-gelatin mixture [4:1, total solid content 12.5% (w/v)] are

5

demonstrated in Figure 2. The gelation temperature of PU1-gelatin mixture is around

6

28°C [Figure 2(a)]. By the dynamic strain sweep test at 25 °C (1 Hz frequency and

7

02500 % oscillatory strain), the gel-to-sol transition of PU1-gelatin occurred at

8

~189% strain [Figure 2(b)]. Dynamic shear results at 25 °C (1% oscillatory strain and

9

0100 Hz) revealed that the complex viscosity of PU1-gelatin decreased as the

10

frequency increased, and was 1.7 Pas at 100 Hz [Figure 2(c)]. In the static shear

11

experiment (03000 s-1), PU1-gelatin showed strong shear thinning effect, with a

12

steady shear viscosity of 0.14 Pas at 100 s-1 shear rate [(Figure S4(a)]. The shear

13

thinning behavior was quantified by power law, where the value of the power law

14

index n  1 represents the shear thinning behavior, n1 indicates the Newtonian fluid,

15

and n  1 denotes the shear thickening behavior. The values of n for the PU1-gelatin

16

bioink and the pure gelatin solution were -0.081 and 0.049, respectively [(Figure S4(a

17

and b)]. Therefore, both of the PU dispersion and pure gelatin solution are shear

18

thinning fluids. When the gelatin solution was mixed with PU dispersion, the PU1-

19

gelatin bioink showed more shear thinning behavior. In Figure 2(b), the G’ value of 17 ACS Paragon Plus Environment


1

the PU1-gelatin bioink dropped slowly until the oscillatory shear strain reached 189%,

2

and afterwards dropped quickly. This finding indicated that PU1-gelatin bioink

3

underwent gel-to-sol transition upon the excessive strain. On the basis of the strain

4

sweep data, we performed creep and creep recovery tests of PU1-gelatin bioink

5

following the protocol in literature42. From the creep recovery results, we recognized

6

that the PU1-gelatin bioink was able to recover quickly after shear removal, and was

7

also strong enough to resist the load at a yield shear stress of ~100 Pa [Figure 2(d)].

8 9

3.3. Mechanical stability and cytocompatibility of PU-gelatin constructs

10

The PU1-gelatin hydrogel ink was printed at room temperature onto the platform set

11

at 2025 °C (Video S1). The printed hydrogel fast recovered its mechanical strength

12

so it could be immediately moved with tweezers and placed in the ionic solution for

13

treatment. The influence of the concentration of CaCl2 on the mechanical properties

14

of PU1-gelatin bioink is illustrated in Figure S6. The storage modulus of PU1-gelatin

15

bioink decreased from 3.7±0.2 kPa to 0.9±0.1 kPa with the increased concentration of

16

CaCl2. The biodegradation profiles of PU1-gelatin and PU3-gelatin constructs are

17

shown in Figure S7(a and b). The one-day remaining weights of PU1-gelatin

18

constructs previously chelated by 0.05, 0.1, 0.2, 0.4, and 0.8 N CaCl2 solution were

19

94.2±1.4%, 96.5±1.3%, 95.3±1.4%, 88.3±1.3%, and 77.6±1.1%, respectively. The 18 ACS Paragon Plus Environment

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1

remaining weights of PU3-gelatin constructs previously chelated by 0.05, 0.1, 0.2, 0.4,

2

and 0.8 N CaCl2 solution were 94.7±1.4%, 97.2±1.4%, 94.4±1.4%, 86.1±1.2% and

3

79.3±1.3%, respectively. The PU1-gelatin or PU3-gelatin constructs chelated by 0.8

4

N (or 0.4 N) CaCl2 showed a relativety lower remaining weight, suggesting that most

5

of the gelatin in the constructs was lost. Constructs chelated with 0.05, 0.1, or 0.2 N

6

CaCl2 solution owned a similar remaining weight. The swelling properties of PU-

7

gelatin constructs are shown in Figure S8(a and b). The one-day swelling ratios of

8

PU1-gelatin constructs previously chelated by 0.05, 0.1, 0.2, 0.4, and 0.8 N CaCl2

9

solution were 352.1±14.9%, 270.1±11.5%, 265.1±11.2%, 251.3±11.5% and

10

122.7±5.2% at 24 h, respectively. The swelling ratio of PU3-gelatin constructs

11

chelated at 0.05, 0.1, 0.2, 0.4 and 0.8 N CaCl2 solution were 271.3.1±11.5%,

12

253.7±10.8%, 254.2±11.2%, 253.3±10.0% and 124.7±5.3% at 24 h, separately. These

13

results revealed that constructs chelated by the 0.05, 0.1 or 0.2 N CaCl2 had good

14

swelling behavior, suggesting water retention in the network structure formed after

15

Ca2+ chelation. Constructs chelated by 0.4 or 0.8 N CaCl2 had low swelling ratios

16

after 3 h probably because of the lack of stable network formation between PU and

17

gelatin under such conditions. The effect of treatment time (5, 10, 15, 20, and 25 min

18

at 25 °C) in 0.2 N CaCl2 solution on the gross structure stability of PU1-gelatin

19

hydrogel at 37 °C is displayed in Figure 3(a). The stability of the structure at 37 °C 19 ACS Paragon Plus Environment


1

increased with the CaCl2 treatment time from 0 to 15 min, while did not further

2

increase when the treatment time was over 15 min. Hydrogels treated for 15 min or

3

longer remained stable at 37 °C without disintegration. The CaCl2 treatment did not

4

adversely affect the survival and health condition of MSCs until 25 min [Figure 3(b)

5

and Figure S9(a)]. As shown in Figure S9 (b and c), the live/ dead images of cells in

6

the PU1-gelatin hydrogel are represented in blue (VB-48 staining) and red (PI

7

staining), respectively. The quantitative data of live cells in the culture medium and

8

PU1-gelatin hydrogel with pretreatment of CaCl2 for 15 min were 93.2% and 92.7%,

9

respectively. The PU1-gelatin hydrogel with pretreatment of CaCl2 for 15 min

10

exhibits good cytocompatibility and is suitable for carrying cells. The stability of the

11

double network was confirmed by static compression [Figure 3(c)]. At 25 °C, the

12

static compressive modulus, strength, and deformation of PU1-gelatin hydrogels were

13

0.60.1 kPa, 1.30.2 kPa, and 62.34.2% before the CaCl2 treatment, and 1.50.2 kPa,

14

and 5.2 kPa, and 69.66.1% after the CaCl2 treatment, respectively. At 37 °C, the

15

above parameters were 2.60.3 kPa, 8.11.1 kPa and 68.88.3%. The double network

16

hydrogel was compressible as well as highly stretchable. The static tensile modulus,

17

strength, and elongation of PU1-gelatin hydrogels were 7.2 0.8 kPa, 3.20.3 kPa and

18

94.110.3% before incubation, and were 14.71.3 kPa, 7.30.6 kPa, and 10510.8%

19

after 24 h of incubation [Figure S10(a)]. The modulus of the double network was 20 ACS Paragon Plus Environment

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1

tunable, e.g. for PU3-gelatin with different solid contents (10, 15, 20, and 25 wt%) at

2

37 °C, the compressive modulus of PU3-gelatin increased from 0.2 kPa to 2.4 kPa

3

[Figure S10(b)]. The dynamic compressive moduli of PU1-gelatin hydrogel after

4

CaCl2 treatment measured immediately at 25 °C or 37 °C without further incubation

5

were 1.60.1 and 2.20.2 kPa, respectively. Incubation in 37 °C PBS enhanced the

6

compressive modulus further to 3.30.2 kPa due to the thermosensitivity of PU1

7

[Figure 3(d)].Therefore, the PU-gelatin hydrogels are deformable and exhibit tunable

8

rigidity/mechanical properties.

9 10

3.4. Higher resolution microextrusion-based bioprinting and long-term

11

cytocompatability of PU-gelatin bioink

12

Three different nozzles (80 m needle-like nozzle, 200 m cone-like nozzle , or 320

13

m micropipette tip) were used in the study. High resolution bioprinting possibility

14

was verified with a nozzle diameter 80 m [Figure 4(a) and Video S2]. PKH26

15

fluorescence staining revealed that MSCs were alive and actively migrated during the

16

10 day culture period [Figure 4(b) and Video S3]. The live/ dead cell images and the

17

cell viability data of PU1-gelatin hydrogel constructs printed through different nozzles

18

(80 m needle-like nozzle, 200 m needle-like nozzle, 200 m cone-like nozzle, and

19

320 m micropipette tip) are shown in Figure S11 (a-e). All groups of PU1-gelatin 21 ACS Paragon Plus Environment


1

hydrogel constructs printed by different nozzles showed live cells in the majority.

2

Meanwhile, the number of dead cells increased slightly as the diameter of nozzle

3

decreased. The cell viability of PU1-gelatin hydrogel constructs printed by the 80 m

4

needle-like nozzle, 200 m needle-like nozzle, 200 m cone-like nozzle, and 320 m

5

micropipette tip were 1304.6%, 1686.3%, 1796.7%, and 1837.8%, respectively.

6

The quantitative data of cell viability in PU1-gelatin hydrogel constructs [Figure 4(c)]

7

indicated that MSCs in PU1-gelatin constructs printed through the 320 m

8

micropipette tip and through the 200 m cone-like nozzle had similar cell

9

proliferation ratios. MSCs in PU1-gelatin printed through the 80 m needle-like

10

nozzle kept growing in 3 days. The printed constructs had maximum MSCs after 3

11

days, which were ~344%, ~337% and ~242% for those through the 320 m tip, 200

12

m nozzle, and 80 m nozzle, respectively. For the ink containing induction agent

13

Y27632 in situ, MSCs in PU1-gelatin constructs underwent chondrogenesis after 10

14

days [Figure 4(d) and Figure S12], where the expression of chondrogenic marker

15

genes (Sox 9, Agg, and Col II) was significantly upregulated. Besides, the gene

16

expressions of the fibrocartilage marker Col I and hypertrophic cartilage marker Col

17

X were low. MSCs in PU1-gelatin constructs with in situ Y27632 had a similar level

18

of chondrogenic differentiation as those with exogenous TGF-3 induction.

19 22 ACS Paragon Plus Environment

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1

4. Discussion

2

Hydrogel ink is the major materials used for 3D bioprinting. Most literature focused

3

on photocrosslinkable hydrogels because of the greater mechanical integrity,

4

printability, and possibility to stack the desired shape with layer-by-layer

5

deposition.5,33,47,48 However, the free radicals produced not only affect the hydrogel

6

viscoelasticity but also damage the activity of embedded cells.12,17 Compared to

7

photoresponsive hydrogels, thermoresponsive hydrogels do not produce free radicals

8

that can affect cell survival. Popular choices of thermoresponsive hydrogels for 3D

9

bioprinting14,26 include Pluronic, argarose, Matrigel, and gelatin. Pluronic hydrogel

10

has good printability and can be gelled above 15 C, but the poor mechanical property

11

and non-degradability have limited its application in bioprinting.49 Argarose and

12

Matrigel are mechanically stronger than Pluronic gel, but have the problem of

13

unsatisfactory printability and poor resolution.50 Gelatin forms hydrogel below 28 C,

14

but cannot remain the hydrogel state at 37 C, and therefore, the UV-sensitive

15

crosslinking moiety is often introduced for gelation and structure stability of gelatin at

16

37 C or otherwise the gelatin must be crosslinked by chemical crosslinkers that are

17

often toxic.51 The mechanical properties, printability, and degradation of

18

thermoresponsive hydrogels may be limited under the chemistry selected,14,52 and the

19

gelling process is relatively slow. It is difficult for one hydrogel to simultaneously 23 ACS Paragon Plus Environment


1

possess good printability, mechanical properties, degradation, and a supportive

2

microenvironment for cell survival and proliferation. The PU-gelatin composite

3

bioinks developed in this study, which possess the advantages of ionic sensitivity

4

hydrogel and thermoresponsive hydrogel and further improve their shortcomings. PU-

5

gelatin bioink do not suffer free radical or toxic crosslinkers, and they have excellent

6

printability, long printing window, as well as tunable mechanical properties and

7

degradation rates that provide a conducive microenvironment for cell growth.

8

The design for the PU-gelatin bioink is summerized in Figure 5 (a and b). We mixed

9

the biodegradable waterborne PU and gelatin in 80/20 weight ratio. PU is the main

10

component in the PU-gelatin bioink, which offers good elasticity, tunable mechanical

11

properties and degradation rates, anti-inflammation, and shear thinning behavior. PU

12

contributes most of the mechanical and degradation properties in the bioink. However,

13

PU has low viscosity at the sol state, which cannot be printed by itself at the sol state.

14

The role of gelatin is to increase the viscosity of PU-gelatin bioink and to provide a

15

temporary fixation effect for the PU-gelatin bioink deposited on the platform. PU

16

dispersion and gelatin mixed in the ratio of 80/20 can have a balance between the

17

temporary fixability and viscosity. If the ratio of PU dispersion in the PU-gelatin

18

bioink is higher than 80 wt%, the structure of PU-gelatin construct will not be well

19

kept on the platform due to the weak gelation and fixation by gelatin. On the other 24 ACS Paragon Plus Environment

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1

hand, if the ratio of gelatin is higher than 80 wt% in the bioink, the viscosity and

2

storage modulus of the bioink will be too high to be printed. Moreover, the chelation

3

effect of gelatin will not be enough to maintain the printed structure at 37C.

4

Meanwhile, the degradation properties of PU-gelatin hydrogel shows that the PU-

5

gelatin hydrogel treated with 0.8 N CaCl2 solution showed the lowest remaining

6

weight ratio. The osmotic pressures of PU-gelatin hydrogel and 0.8 N CaCl2 solution

7

were 300 and 1200 mOsm, respectively. The high osmotic pressure of 0.8 N CaCl2

8

solution may prevent the effective diffusion of ion into PU-gelatin hydrogel to form

9

the Ca2+-chelated network. The gelatin in such PU-gelatin hydrogel would transform

10

to the sol state at 37 oC. This probably explained why 0.8 N CaCl2 solution did not

11

achieve a better fixation effect than 0.2 N CaCl2 in treating PU-gelatin hydrogel. After

12

optimization, we selected the specific weight ratio of 80/20 for PU dispersion and

13

gelatin to balance the printing and fixation, followed with immersion of the structure

14

in 0.2 N CaCl2 for chelation. The compressive modulus of PU1-gelatin constructs

15

were 3.3±0.2 kPa at 37C, which was considered to promote chondrocyte growth.53

16

Besides, we mixed PU3 and gelatin in 80/20 ratio and further adjusted the total solid

17

contents of PU3-gelatin bioink. The data showed that the compressive modulus of

18

PU3-gelatin hydrogel was increased by increasing the solid contents. PUs had

19

different chemical compositions and distinct degradation rates (PU3 >PU1 >PU2).38 25 ACS Paragon Plus Environment


1

Therefore, the mechanical properties and degradation rates of PU-gelatin bioink can

2

be tuned by changing PU composition or regulating the solid content/ratio of PU and

3

gelatin.

4

The PU-gelatin hydrogel bioink has excellent printability and can print the

5

complicated structure of an organ or tissue such as nose. The printed constructs have

6

good elasticity and mechanical strength. Although many natural materials are

7

sensitive to Ca2+, the Ca2+-induced gelation is usually too fast for printing. In this

8

study, we observed that the divalent cations such as the Ca2+ and Ba2+ could promote

9

the gelation of PU dispersion and stabilization at 37 C. However, the effect of

10

divalent cations on gelatin only was not as obvious, i.e. gelatin alone treated with Ca2+

11

was still fluid-like at 37 C. The chelation reation of Ca2+ that occurred to stablize the

12

PU-gelatin double network was supproted by the shift of the NH group associated

13

bands to higher wavenumbers (red shift) in IR spectra.54-57 The shift was attributed to

14

the coordination bonding between the divalent cation and a lone pair electron of NH

15

groups after the CaCl2 treatment. Regarding the double network forming mechanism,

16

the rich hydrophilic functional group on PU NP surface could chelate with divalent

17

cations.58 The functional groups of PU to interact with divalent cations were more

18

abundant than those of gelatin, and thus Ca2+ had a stronger gelation effect on PU.

19

Meanwhile, gelatin molecules comprise COOH, NH2, and CONH2 groups,59 and 26 ACS Paragon Plus Environment

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1

chelation of gelatin by Ca2+ may still occur to stabilize the network.60 We thus suggest

2

a two-stage double network formation mechanism for PU-gelatin (Figure 6). When

3

the PU-gelatin hybrid is treated with Ca2+ solution, each molecule could chelate with

4

Ca2+ to form the chemical network, with proper mechanical properties maintained at

5

37 C. PU may undergo slow self-assembly, where the secondary force slowly

6

increases with time at 37 C.61 The so-formed PU physical network further increases

7

the strength of the hybrid hydrogel. The PU-gelatin hybrid hydrogel is “konjac-

8

like“ and can be picked up by hands. In literature, alginate also forms network with

9

Ca2+, but the printability, dimensional stability, and mechanical strength are relatively

10

poor and the in vivo degradation is often too fast, which limits the clinical

11

applications.15,62,63 Cells should be cultured in an isotonic solution (~300 mOsm) to

12

avoid cell crenation and lysis

13

crenate in a hypertonic solution. The osmotic pressure of PU-gelatin bioink is the

14

same as that of cell culture medium (~ 300 mOsm). The osmotic pressure of 0.05, 0.1,

15

0.2, 0.4, and 0.8 N CaCl2 solution is 75, 150, 300, 600, and 1200 mOsm, respectively.

16

The osmotic pressure of 0.2 N CaCl2 solution is similar to the cell culture medium,

17

and the calcium ion may diffuse into the PU-gelatin bioink without the osmotic

18

pressure change. Moreover, cells in the PU-gelatin bioink would not crenate or lyse in

19

the chelation process with the 0.2 N CaCl2 solution.

64.

Cells may lyse in a hypotonic solution, and may



Page 28 of 49

1

The rheological properties of bioink are very important for printability. The ink

2

must have shear thinning properties to be extruded through the nozzle.33 A large shear

3

force/strain is generated during the process.65 From the dynamic frequency sweep, the

4

complex viscosity of PU1-gelatin hydrogel decreased almost linearly with the

5

frequency in log-log scale. The static shear experiment also revealed the same

6

tendency. The excellent shear thinning properties are probably associated with the

7

unique viscoelasticity of PU NPs. Regarding the rheology suitable for printing, the

8

more negative slope of viscosity vs. shear rate log-log plot (more shear thinning) may

9

facilitate printing. In this study, the slope of PU1-gelation was 1.2, which was more

10

negative than that of silk-based bioink (slope ~ 0.2)

11

(slope ~ 0.6).67 The latter two had printability but the stacking ability and structure

12

stability were not as satisfactory. Certain Pluronic-diacrylate bioink (slope ~ 0.5)

13

and silk fibroin-gelatin bioink (slope ~ 1.9)

14

ability but the former had to be photocrosslinked.

67

66

and alginate-based bioink

12

also had good printability/stacking

15

Although bioprinting is facilitated by shear thinning, the structure stability of

16

printed hydrogel relies on the “fast recovery” of gel structure. Owing to the good

17

shear thinning properties, the ink in our study became sol-like upon a high shear force

18

when passing through the gradually narrow area of the nozzle. After printing, the

19

shear force was removed and the network structure of PU-gelatin hydrogel recovered 28 ACS Paragon Plus Environment

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1

quickly. In literature, the fast gelation to a strength of above 250 Pa is necessary for

2

layer-by-layer stacking.8,10-12,67,68 PU1-gelatin bioink after printing could have the

3

strength recovered to ~400 Pa, thereby facilitating the layer-by-layer deposition.

4

Besides, the tan δ value was 0.18 for PU1-gelatin bioink, similar to that of the silk

5

fibroin–gelatin bioink (tan  ~0.16)

6

bioink (tan  ~0.1).12 The tan  value of PU1-gelatin is in a better range of

7

viscoelasticity and damping ability. Moreover, the proper rheological property allows

8

the gel network of PU1-gelation to be destroyed only when the strain is over 189%.

9

Meanwhile, silk fibroin–gelatin bioink was

destroyed,

and

67

and greater than that of the Pluronic-based

67

had a critical strain at 100% where the

methacrylated

hyaluronic

acid

(MeHA)

68

10

network

11

photocrosskable system only endured 30% strain. The PU-gelatin ink gelled

12

immediately, allowing layer-by-layer deposition into a large construct. The structure

13

stability (enduring large strain) and elasticity (fast recovery) of the gel network of PU-

14

gelatin allowed the printed constructs to be picked up and squeezed by hand.

15

Microextrusion-based bioprinting usually has a limit of resolution 100200

16

m.31,69,70 Most types of the bioink are very difficult to pass through

Double network polyurethane-gelatin hydrogel with tunable modulus

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