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Norbornene-modified poly(glycerol sebacate) as a

Cite this: DOI: 10.1039/c7py00323d
photocurable and biodegradable elastomer†
Yi-Cheun Yeh,a Liliang Ouyang,a,b Christopher B. Highleya and Jason A. Burdick *a

Poly(glycerol sebacate) (PGS) is a biodegradable elastomer that has emerged as a promising material in
biomedical applications; however, the traditional thermal crosslinking of PGS limits its processing in the
fabrication of three-dimensional (3D) scaffolds. Here, we designed a new photocurable PGS that uses
thiol–ene click chemistry to control PGS crosslinking. Specifically, norbornene-functionalized PGS (Nor-
PGS) macromers were crosslinked by varied amounts of a 4-arm thiolated crosslinker to program pro-
perties such as mechanics and degradation. The Nor-PGS crosslinked rapidly (<1 min to plateau modulus)
under ultraviolet light and the storage modulus was dependent on the crosslinker amount, with a
maximum storage modulus observed with an equimolar norbornene/thiol ratio. Tensile properties of
Nor-PGS were also altered by the crosslinker concentration, with a simultaneous increase in elastic
modulus and failure stress and decrease in elongation (ranging from ∼170–240%) with higher crosslinker
concentrations. In turn, the degradation of Nor-PGS was faster with lower crosslinker concentrations. To
illustrate the processing of Nor-PGS, porous scaffolds with a range of sizes and shapes were fabricated
Received 26th February 2017, using extrusion-based 3D printing, where the viscous macromer was extruded and crosslinked with con-
Accepted 27th March 2017
tinuous ultraviolet light exposure. Printed scaffolds supported the culture and proliferation of fibroblasts.
DOI: 10.1039/c7py00323d These results indicate that crosslinked Nor-PGS is a promising functional elastomer for the fabrication of
rsc.li/polymers property-controllable scaffolds for biomedical applications.

Introduction tissue engineering applications (e.g., cardiac,13,14 vascular,15,16

neural17 tissues).
Poly(glycerol sebacate) (PGS), a thermally-crosslinked poly- For use in biomedical applications, such as in the fabrica-
ester, has great potential in tissue engineering due to its elas- tion of tissue engineering scaffolds or as injectable materials,
ticity, biodegradability and cytocompatibility.1–3 PGS is pre- PGS must undergo a controlled crosslinking reaction.
pared through a polycondensation reaction of glycerol and However, the traditional crosslinking of PGS is restricted by
sebacic acid, where the mechanical properties and degradation the difficulties of curing PGS at high temperatures and under
kinetics of PGS can be tailored by varying the curing tempera- vacuum. To expand the processing capabilities of PGS beyond
ture and time to match the requirements of intended appli- thermal crosslinking, additional chemical modifications can
cations.4,5 For biological studies, PGS provides a conducive be used. Towards this, PGS has been functionalized with
surface for cell adhesion and growth.6,7 PGS degrades in a con- various reactive groups (e.g., acrylates18,19 and cinnamates20)
trolled manner with degradation products that are well toler- to allow crosslinking at room temperature using light.
ated in biological systems.1,8 Particularly, PGS possesses Acrylated-PGS (Acr-PGS) undergoes a free-radical photo-
rubber-like elasticity that can sustain and recover from defor- initiated polymerization that crosslinks in the presence of a
mation to match the elastomeric properties of many soft photoinitiator and corresponding light, whereas cinnamate-
tissues in the body.9,10 With these unique properties and posi- modified PGS (PGS-CinA) undergoes a photodimerization
tive attributes, PGS is increasingly being studied as scaffolds upon irradiation with ultraviolet light.
in dynamic environments (e.g., heart11 and cartilage12) and for PGS formed from Acr-PGS exhibits tunable mechanical pro-
perties by controlling the degree of acrylate substitution and
PGS molecular weight.18,19 By exploiting the photocrosslinking
a
Department of Bioengineering, University of Pennsylvania, Philadelphia, mechanism, Acr-PGS has been processed through electro-
Pennsylvania, USA. E-mail: burdick2@seas.upenn.edu spinning to fabricate biodegradable fibrous scaffolds with
b
Department of Mechanical Engineering, Tsinghua University, Beijing,
diverse mechanics (∼60 kPa to 1 MPa).21 Towards complex
People’s Republic of China
† Electronic supplementary information (ESI) available. See DOI: 10.1039/ structures, Acr-PGS has also been applied to generate macro-
c7py00323d porous tissue scaffolds (i.e., ear and meniscus) through 3D

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printing.22 PGS-CinA forms networks where the properties 5-Norbornene-2-carbonyl chloride was prepared by reacting
(e.g., mechanics and degradation) are controlled through the a mixture of 5-norbornene-2-carboxylic acid (0.58 ml,
degree of substitution.20 Likewise, elastomers from PGS-CinA 4.75 mmol, a mixture of endo- and exo-, predominantly endo),
have been formed that are cytocompatible and can be pro- oxalyl chloride (0.6 ml, 7.13 mmol) and dimethylformamide
cessed using replica-molding techniques.20 In addition, (DMF, 75 µl, Thermo Fisher Scientific), where DMF was used
PGS-CinA has been utilized to generate mesoporous elasto- as a catalyst. These reagents were mixed and stirred in anhy-
meric matrices to release small molecules in a controlled drous dichloromethane (DCM, 10 ml, Thermo Fisher
manner.23 Scientific) at room temperature for 6 h under N2. The excess
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Here, we introduce a new approach to crosslink modified oxalyl chloride and solvent were removed under reduced
PGS with photoinitiated thiol–ene reactions, through the pressure. 5-Norbornene-2-carbonyl chloride was re-dissolved in
modification of PGS with norbornene moieties. The thiol–ene anhydrous DCM and directly used for the functionalization of
reaction is a stepwise reaction that occurs in a stoichiometric PGS.
ratio24 and is also less susceptible to oxygen inhibition than PGS prepolymer (5 g) was dissolved in anhydrous DCM
other photoinitiated reactions.25 Thus, thiol–ene reactions are (60 ml) containing 500 ppm 4-methoxyphenol and 0.1 wt%
able to rapidly polymerize at lower concentrations of photo- 4-(dimethylamino)pyridine (DMAP). The reaction flask was
initiator and under ambient conditions,26 which may further cooled to 0 °C and the solution was purged with N2 for
broaden the processing techniques available. In particular, 10 min. 5-Norbornene-2-carbonyl chloride (4.75 mmol in 7 ml
due to the oxygen tolerance, thiol–ene reactions are highly DCM) was added dropwise into the PGS solution parallel to tri-
cytocompatible as they do not generate intracellular reactive ethylamine (1 ml, 7.13 mmol) and then stirred at room temp-
oxygen species (ROS) that are usually observed in common erature overnight (Fig. 1a, bottom). An additional 500 ppm
radical polymerizations.27,28 Additionally, the step-growth 4-methoxyphenol was added to the reaction solution and DCM
mechanism results in uniform and controlled networks,29 was removed using a rotary evaporator. Ethyl acetate was used
which may be advantageous compared to the heterogeneous to dissolve the remaining viscous liquid and the solution was
network structure from chain-growth polymerization. Finally, vacuum filtered to remove triethylamine salts. Ethyl acetate
thiol–ene reactions can be conducted under cytocompatible was removed using a rotary evaporator to leave a viscous
doses of ultraviolet light (e.g., 10 mW cm−2), providing an liquid. The viscous liquid was then dissolved in DCM and
advantage over the cinnamate-based photocrosslinking that washed with hydrochloric acid (10 mM, three times), water
requires high energy input (e.g., intense ultraviolet light (two times), and dried with anhydrous magnesium sulfate.
(>100 mW cm−2) or long exposure time (∼2–3 h)) to achieve After filtration of magnesium sulfate, DCM was removed via
effective dimerization.20,23,30,31 rotovapping to leave a viscous liquid, which was further dried
Norbornene-functionalized PGS (Nor-PGS) is crosslinked by in an oven (37 °C) overnight, and then purged with N2 and
thiolated molecules in the presence of light and photoinitiator. stored at −20 °C. The norbornene modification of Nor-PGS
The mechanical properties and degradation rates of Nor-PGS was ∼15% (calculation was based on the NMR analysis as
can be fine-tuned through changes in the amount of cross- described below) and the yield of Nor-PGS was ∼96% (4.8 g as
linker, enabling the rational design of desired properties for final product). To alter the norbornene modification on Nor-
specific applications. We applied the photocrosslinking of PGS, the PGS prepolymer was changed to 6.5 g and 3.5 g for
Nor-PGS macromers and thiolated crosslinkers for the fabrica- 11% and 19% modification, respectively.
tion of macroporous scaffolds using extrusion-based 3D print- The chemical structures of PGS prepolymer and Nor-PGS
ing and investigated their cytocompatibility with fibroblasts macromers were verified using 1H nuclear magnetic resonance
towards utility in tissue engineering. (1H NMR, Bruker Advance 360 MHz, Bruker). The chemical
composition was majorly determined by calculating the signal
integrals of –COCH̲2CH̲2CH̲2– at 1.31, 1.69–1.55, and
Experimental 2.41–2.27 ppm for sebacic acid, –CH̲2CH̲– at 3.5–5.2 ppm for
glycerol, and –CH̲vCH̲– at 6.26–5.88 ppm for the vinyl protons
Materials
on the norbornene groups. The signal intensity of the methyl-
All chemicals were purchased from Sigma-Aldrich and used as ene groups of sebacic acid (1.69–1.55 ppm) and the vinyl
received unless otherwise indicated. protons on the norbornene groups (signal intensities of
6.26–5.88 ppm) were used to calculate the extent of norbor-
Synthesis and characterization of PGS prepolymer and Nor- nene modification. In detail, the signal intensity of the methyl-
PGS ene groups of sebacic acid was normalized to 4, and the signal
PGS prepolymer was synthesized via the condensation reaction intensity of vinyl protons on the norbornene groups was
of equimolar amounts of glycerol (Thermo Fisher Scientific) divided by 2 to determine the percentage of norbornene on
and sebacic acid.1 The reagents were mixed and purged with Nor-PGS.
N2 for 10 min before heating. The mixture was stirred at The peak assignments in the NMR spectra for Nor-PGS
120 °C under N2 flow for 2 h and then a vacuum of 12 mbar macromers (15% norbornene modification) are listed below.
was applied for 46 h to obtain PGS prepolymer (Fig. 1a, top). 1
H NMR (360 MHz, CDCl3) δ/ppm: 1.31 (52H, s, –CH̲2–),

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Fig. 1 (a) Synthesis scheme of PGS and Nor-PGS, with the synthesis of Nor-PGS from PGS through the coupling of 5-norbornene-2-carbonyl
chloride to hydroxyl groups on PGS. (b) 1H NMR spectrum of Nor-PGS.

1.51–1.23 ppm (2H, m, bridgehead proton and ring protons on acetophenone (DMPA, 0.1 wt%) for crosslinking. Dynamic
the norbornene), 1.69–1.55 (26H, d, –CH̲2CH2O(CO)–), oscillatory time sweeps were performed using an AR2000
2.23–1.86 ppm (2H, m, bridgehead proton and ring protons on stress-controlled rheometer (TA Instruments) with an ultra-
the norbornene), 2.41–2.27 (26H, m, –CH̲2O(CO)–), 3.30–2.90 violet light-guide accessory (SmartSwap™, TA Instruments)
(3H, m, bridge and α protons on the norbornene), 3.50–5.32 connected to an ultraviolet light source (Omnicure S1000,
(36H, m, ORCH̲2CH̲O–, R = H, norbornene or polymer chain + EXFO). The photocrosslinking of Nor-PGS was carried out
norbornene), 6.26–5.88 (2H, m, vinyl protons on the under exposure to ultraviolet light (365 nm, 10 mW cm−2).
norbornene). Storage (G′) and loss (G″) moduli with time were monitored
The molecular weight of the PGS prepolymer was measured under 0.5% strain and 1 Hz, using a cone and plate geometry
using gel permeation chromatography (GPC). PGS prepolymer (59 min 42 s (0.995°) cone angle, 20 mm diameter, 27 μm gap)
was dissolved in tetrahydrofuran (THF) at a concentration of at 25 °C.
5 mg mL−1. GPC-RI analyses were performed in THF (1.0 mL
min−1, 30 °C) using a Viscotek GPCmax with a VE 2001 GPC Tensile testing
solvent/sample module, a 2600 UV-PDA detector, and a TDA Photocured Nor-PGS samples with varied thiol/norbornene
305 triple detector array. A set of two columns, including one ratios (N = 0.5, 0.75 and 1) were prepared as dog-bone shaped
PLgel 5 µm mixed-D and one PLgel 5 µm mixed-C columns, specimens (∼1 mm height, overall length of 30 mm, and a nar-
was used for separation and poly(methyl methacrylate) rowed section that was 10 mm long and 5 mm wide). Samples
EasiVials standards (Agilent Technologies) were used for were loaded into textured grips on an Instron 5848 universal
calibration. testing system (Instron Corp., 5 N load cell) and tested with a
0.01 N preload and uniaxial extension (10 mm min−1) until
Rheological measurement failure. Force and displacement data were acquired during
Nor-PGS macromers were mixed with pentaerythritol tetrakis loading and analyzed computationally (MATLAB, MathWorks)
(3-mercaptopropionate) (PETMP) and 2,2-dimethoxy-2-phenyl- to determine sample stresses (calculated as measured forces

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divided by cross-sectional areas), failure stresses (the Hamiliton Company) with affixed blunt tip 25 G needles that
maximum stresses achieved), failure strains (the strains at were 6 mm long. Syringes were loaded onto the 3D printer and
which the failure stresses were achieved), and elastic moduli the printing was performed at ambient temperature (approxi-
(measured as the slopes from 40% to 50% strains). Cyclical mately 23 °C) with continuous irradiation of the print area with
loading test of Nor-PGS sample (N = 1) was performed at a jog ultraviolet light (365 nm, 10 mW cm−2) during printing and for
rate of 60 mm min−1 in the elongation range of 30%–60% five minutes post-printing. Printed PGS scaffolds were imaged
during 50 consecutive cycles. using scanning electron microscopy (SEM, FEI Quanta 600
environmental scanning electron microscope).
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Degradation behavior
For degradation analysis, Nor-PGS polymer disks (1 mm thick, Cell culture and characterization of cell-seeded PGS scaffolds
2 mm diameter) were prepared with different thiol/norbornene NIH 3T3 fibroblast cells (ATCC) were cultured at 37 °C under a
ratios (N = 0.5, 0.75 and 1) as described above. PGS disks were humidified atmosphere of 5% CO2 in high glucose Dulbecco’s
weighed, incubated in 1.5 ml of phosphate-buffered saline (PBS, Modified Eagle’s Medium (DMEM, 4.5 g L−1 glucose) contain-
pH 7.4), and placed on an orbital shaker at 37 °C. The solution ing 10% fetal bovine serum (FBS) and 1% penicillin/streptomy-
was replaced with fresh PBS weekly. At each time point, three cin. For cell seeding, 3D-printed PGS scaffolds were sterilized
samples of each scaffold type were removed, lyophilized by incubation in 70% ethanol for 30 min, washed with PBS,
(Freezone 4.5, Labconco), and weighed to determine mass loss. and incubated in serum-contained media for 24 h. As an
initial test of cell adhesion and viability, 3T3 cell suspensions
3D printing and imaging (1 × 106 cells in 2 ml) were added onto 3D printed PGS
Printing used a 3D printer adapted for deposition of materials scaffolds (2-layer, 10 × 10 mm, height: 0.16 mm) in a cell
by extrusion, which was described previously.22,32,33 3D struc- culture dish (diameter × height, 35 mm × 10 mm). After 24 h,
tures (cube, nose and ear) were printed from respective 3D com- the cell-seeded scaffolds were transferred to a new cell culture
puter-aided design (CAD) models. Nor-PGS macromers, PETMP dish and stained with calcein-AM (2 µM in PBS, Life
(thiol/norbornene = 1) and DMPA (0.1 wt%) were mixed in DCM Technologies) at room temperature for 20 min before imaging.
and dried in oven (37 °C) overnight before printing. The Fluorescent images were taken using an Olympus
materials were loaded into glass syringes (Gastight syringes, BX51 microscope (B&B Microscopes Limited).

Fig. 2 (a) Scheme of Nor-PGS crosslinking through the light initiated thiol–norbornene reaction between Nor-PGS and PETMP. (b) Rheology of the
photocrosslinking of Nor-PGS in the presence of PETMP and ultraviolet light (365 nm, 10 mW cm−2). The storage moduli (G’) of Nor-PGS networks
were varied through (c) the extent of norbornene modification (11, 15, 19% with thiol/norbornene = 1) and (d, e) thiol/norbornene ratio (N) with 15%
norbornene modified Nor-PGS.

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To assess cell proliferation, 3T3 cell suspensions (1 × 105 Functionalization of PGS, such as by acrylation,18,19 has been
cells in 100 µl) were seeded onto 3D printed PGS scaffolds used to process PGS into complex, multi-scale structures.
(10-layer, 10 × 10 mm, height: 0.8 mm) in a 24-well cell culture Here, we implemented a new modification (i.e., norbornene)
plate. The scaffolds were incubated for 2 h and then 2 ml of of PGS to allow the processing of PGS via thiol–ene cross-
culture medium was added. After 24 h, the scaffolds were linking, to impart features such as rapid polymerization,
transferred to a non-tissue culture treated 12-well plate and oxygen insensitivity, orthogonal reaction between thiols and
assessed for metabolic activity for up to 8 days using an norbornenes, and controllable crosslinking. This modification
Alamar blue assay according to the manufacturer’s protocol may further expand the processing capability of PGS and
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(Invitrogen Biosource, USA). provide a simple technique to tune PGS properties.

PGS prepolymer was synthesized via a polycondensation
Statistical analysis reaction of glycerol and sebacic acid in a 1 : 1 molar ratio and a
One-way analysis of variance (ANOVA) was performed on data weight average molecular weight (Mw) of 7.06 kDa with polydis-
sets to determine statistical significance of differences in persity index of 5.06 was obtained via reaction for 46 h at
mechanical testing, degradation and cell activity. Significance 120 °C (Fig. 1a, top). Hydroxyl groups of PGS were then reacted
was set at p < 0.05 with *, ** or *** indicating p < 0.05, 0.01 or with 5-norbornene-2-carbonyl chloride to obtain norbornene-
0.001, respectively. Error bars are reported in figures as the functionalized PGS (Nor-PGS) (Fig. 1a, bottom). The incorpor-
standard deviation (s.d.) unless otherwise noted. ation of norbornene groups to the PGS prepolymer (11–19%
modification) was confirmed in 1H NMR by the appearance of
peaks at δ 6.26–5.88 ppm for the vinyl protons on the norbor-
Results and discussion nene groups (Fig. 1b).
The crosslinking of Nor-PGS was investigated using photo-
PGS is an elastic, biocompatible and biodegradable polymer rheology. The norbornenes on Nor-PGS macromers undergo a
increasingly used in a variety of biomedical applications. thiol–ene reaction with thiols on the four-arm thiolated cross-

Fig. 3 (a) Representative images of Nor-PGS samples with varied thiol/norbornene ratios (N) before and after (images taken immediately before
failure) tensile loading. (b) Representative tensile stress versus elongation profiles for networks formed from the Nor-PGS macromers with varied
thiol/norbornene ratios. (c) Modulus, (d) failure stress, and (e) failure strain of the Nor-PGS samples with varied thiol/norbornene ratios. n =
3 measurements per group, data presented as mean ± s.d., **p < 0.01 and ***p < 0.001.

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linker PETMP in the presence of the photoinitiator DMPA soft Nor-PGS network with high elongation of over 200%. In
(0.1 wt%) and ultraviolet light (365 nm, 10 mW cm−2) (Fig. 2a). addition, the hysteresis test of Nor-PGS (N = 1) was performed
Nor-PGS macromers were crosslinked by PETMP rapidly as the in the elongation range of 30%–60%, showing that the Nor-
crossover of the storage (G′) and the loss (G″) moduli occurred PGS elastomer maintained its tensile property with minimal
in seconds, and G′ increased multiple orders of magnitude creep deformation after 50 tensile cycles (Fig. 4).
and reached a plateau within one minute of light exposure Previous studies have reported that PGS22,34,35 and the thio-
(Fig. 2b). The rheological properties of Nor-PGS networks can lated crosslinker PETMP36 present hydrolytic degradability due
be altered through the extent of norbornene modification of to the ester moieties in their structures. Here, degradation of
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the Nor-PGS macromers or through the thiol/norbornene ratio (N). Nor-PGS was investigated in PBS ( pH 7.4) at 37 °C (Fig. 5).
The G′ at the plateau increased with increasing norbornene Mass loss of the N = 0.5 sample was greater than the N = 0.75
modification in Nor-PGS, ∼12, 98, and 124 kPa for 11, 15, and N = 1 groups, indicating that decreased crosslinking in
and 19% Nor-PGS modification, respectively (Fig. 2c). Using Nor-PGS results in faster degradation. This control over degra-
the same Nor-PGS (15% norbornene modification), the plateau dation properties in Nor-PGS by varying crosslinker amounts
G′ was controlled with the concentration of PETMP due to would be useful to shorten or extend the lifetime of the Nor-
varied extents of crosslinking and ranged from ∼17 to 98 kPa PGS scaffolds depending on the application.
(Fig. 2d and e). The maximum G′ was observed at a thiol/nor- The processing of Nor-PGS into scaffolds was demonstrated
bornene ratio equal to 1 as each thiol was able to react with a using 3D printing. We applied Nor-PGS as a photocurable ink
norbornene to make an effective crosslink. With a thiol/nor- for 3D printing to fabricate porous structures through the
bornene ratio greater than one, the G′ dropped from the extrusion of material in a layer-by-layer deposition process.
maximum value due to saturation of norbornene groups and Mixtures of Nor-PGS macromers, PETMP crosslinkers and
excess thiols, yielding networks with free pendant thiols.
Additional mechanical properties of Nor-PGS networks
were determined with tensile testing. Nor-PGS samples were
prepared with varied thiol/norbornene ratios (N = 0.5, 0.75 and 1)
to vary the extent of crosslinking. The % elongation of Nor-
PGS was modulated by the PETMP amount used for photocross-
linking, showing ∼170, 200, and 240% elongation for thiol/
norbornene ratios of 1, 0.75, and 0.5, respectively (Fig. 3a and
b). The measured properties of the Nor-PGS samples also
varied with PETMP amount. The highest elastic modulus of
∼400 kPa was observed for the Nor-PGS with N = 1, followed by
the N = 0.75 at ∼340 kPa, and N = 0.5 at ∼110 kPa (Fig. 3c).
Failure stresses presented a similar trend, with N = 1 failing at
∼790 kPa, N = 0.75 at ∼690 kPa, and N = 0.5 at ∼260 kPa
(Fig. 3d). Failure strains followed an opposite trend, and were
measured at ∼170% for N = 1, ∼190% for N = 0.75, and ∼210%
for N = 0.5 (Fig. 3e). All Nor-PGS samples exhibited elastic pro- Fig. 5 Nor-PGS degradation in PBS ( pH 7.4) at 37 °C. In each Nor-PGS
perties, and their tensile properties were easily tuned by chan- (N = 0.5, 0.75 and 1) group, the statistical significance (denoted in gray
asterisk) was calculated based on the samples of week 8 (W8) and week
ging the crosslinker amount. Increased crosslinker amount, up
12 (W12) relative to the sample of week 4 (W4). For comparison between
to N = 1, resulted in a higher extent of crosslinking, presenting groups, the statistical significance is denoted in black and red asterisk.
stronger mechanical properties of the Nor-PGS network, while n = 3 measurements per group, data presented as mean ± s.d.,
decreased crosslinker amounts resulted in the formation of a *p < 0.05, **p < 0.01 and ***p < 0.001.

Fig. 4 (a) Representative images and (b) tensile stress–elongation profile of Nor-PGS (N = 1) during 50 cycles of tensile loading.

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DMPA photoinitiator were prepared as inks to print designed Nor-PGS (Fig. 6a). The open-lattice cube structure was
constructs, where the materials were extruded from a syringe imaged using SEM to illustrate its porous structure and
on a standard 3D printer and cured by continuous exposure to uniform printed filaments (Fig. 6b). In addition, hetero-
ultraviolet light (365 nm, 10 mW cm−2) upon extrusion. A morphic structures were also printed to further illustrate print-
typical multi-layer lattice cube structure was printed with ability, including a human nose and ear of ∼30 mm in length
good integrity (65 layers with 8 × 8 mm in cross-section and (Fig. 6c and d). The printed Nor-PGS structures were elastic, as
5 mm in height), demonstrating the general printability of the ear can be deformed under an applied force and returned
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Fig. 6 (a) 3D printed Nor-PGS lattice cube structure and (b) SEM image. The lattice structure was printed up to 65 layers with 8 × 8 mm in cross-
section, 5 mm in height, and 1 mm for the gap between filaments. Computer designs and printed cartilaginous structures of (c) a nose and (d) an
ear.

Fig. 7 (a) 3T3 fibroblasts cultured on a 2-layered scaffold and stained with calcein-AM after culture for 24 h. (b) Normalized (to day 1 values) meta-
bolic activity of 3T3 fibroblasts on a 10-layered scaffold, where activity was determined for up to 8 days using an Alamar blue assay. n = 3 measure-
ments per group, data presented as mean ± s.d., values at all time points were statistically significant from each other ( p < 0.001).

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to its geometry on removal of that force (Movie S1†). These 5 X. Li, A. T.-L. Hong, N. Naskar and H.-J. Chung,
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To demonstrate the cytocompatibility of crosslinked Nor- Mater. Res., Part A, 2007, 83, 1070–1075.
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(Fig. 7a), which was expected based on previously reported Part A, 2003, 66, 192–197.
observations of cellular adhesion to PGS surfaces.1,2 Cellular 9 M. J. N. Pereira, B. Ouyang, C. A. Sundback, N. Lang,
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