RESEARCH ARTICLE

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Biodegradable Poly(ester) Urethane Acrylate Resins for

Digital Light Processing: From Polymer Synthesis to 3D
Printed Tissue Engineering Constructs
Rong Wang, Febriyani Damanik, Tobias Kuhnt, Armand Jaminon, Shahzad Hafeez,
Hong Liu, Hans Ippel, Pieter J. Dijkstra, Nicole Bouvy, Leon Schurgers, A. Tessa ten Cate,
Aylvin Dias, Lorenzo Moroni,* and Matthew B. Baker*

1. Introduction
Digital light processing (DLP) is an accurate and fast additive manufacturing
technique to produce a variety of products, from patient-customized Over the past decades, additive manufactur-
ing (AM) has emerged as a powerful tech-
biomedical implants to consumer goods. However, DLP’s use in tissue
nology to fabricate biomaterial-based im-
engineering has been hampered due to a lack of biodegradable resin plantable objects with a complex geome-
development. Herein, a library of biodegradable poly(esters) capped with try suitable for tissue regeneration. Among
urethane acrylate (with variations in molecular weight) is investigated as the AM techniques, lithography-based methods
basis for DLP printable resins for tissue engineering. The synthesized like stereolithography (SLA) and DLP, offer
oligomers show good printability and are capable of creating complex unparalleled spatial resolution. With these
high-resolution techniques, it becomes pos-
structures with mechanical moduli close to those of medium-soft tissues
sible to produce well-defined structures
(1–3 MPa). While fabricated films from different molecular weight resins show from photo-polymerizable resins.[1] Scaf-
few differences in surface topology, wettability, and protein adsorption, the folds produced from these techniques can
adhesion and metabolic activity of NCTC clone 929 (L929) cells and human better mimic the complexity of human tis-
dermal fibroblasts (HDFs) are significantly different. Resins from higher sues and offer patient-specific customiza-
tion. Unfortunately, a dearth of well-defined
molecular weight oligomers provide greater cell adhesion and metabolic
and synthetic biodegradable resins severely
activity. Furthermore, these materials show compatibility in a subcutaneous in limits the application of DLP to fabricate
vivo pig model. These customizable, biodegradable, and biocompatible resins scaffolds for regenerative therapies.
show the importance of molecular tuning and open up new possibilities for While various studies have explored suit-
the creation of biocompatible constructs for tissue engineering. able photo-precursor materials used for
biomedical applications,[2] there are only
a few examples of chemically defined and
biodegradable resins for DLP printing.[3]

R. Wang, F. Damanik, T. Kuhnt, S. Hafeez, P. J. Dijkstra, L. Moroni, A. Jaminon, H. Ippel, L. Schurgers

M. B. Baker School for Cardiovascular Diseases
Department of Complex Tissue Regeneration Faculty of Health Medicine and Life Sciences
MERLN Institute for Technology-Inspired Regenerative Medicine Maastricht University
Maastricht University Maastricht 6229 ER, The Netherlands
Maastricht 6229 ER, The Netherlands H. Liu, N. Bouvy
E-mail: l.moroni@maastrichtuniversity.nl; Department of Surgery
m.baker@maastrichtuniversity.nl Maastricht University Medical Center
Maastricht 6229 HX, The Netherlands
A. T. ten Cate
Department of Materials for Additive Manufacturing
TNO
P.O. Box 6235, Eindhoven 5600 HE, The Netherlands
The ORCID identification number(s) for the author(s) of this article
can be found under https://doi.org/10.1002/adhm.202202648 A. T. ten Cate
Department of Additive Manufacturing
© 2023 The Authors. Advanced Healthcare Materials published by
Brightlands Materials Center
Wiley-VCH GmbH. This is an open access article under the terms of the Urmonderbaan 22, Geleen 6167 RD, The Netherlands
Creative Commons Attribution-NonCommercial License, which permits
use, distribution and reproduction in any medium, provided the original A. Dias
work is properly cited and is not used for commercial purposes. DSM Biomedical
DSM
DOI: 10.1002/adhm.202202648 Koestraat 1, Geleen 6167 RA, The Netherlands

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Scheme 1. Synthesis of biodegradable poly(𝜖-caprolactone-co-d,l-lactide) urethane acrylate macromers enabled the formulation of biodegradable resins
useful for DLP fabrication and processing. 3D objects were fabricated, and the biocompatibility and cellular adhesion of the resulting constructs showed
their suitability for use as tissue engineering scaffolds.
Natural polymers can also be used for DLP printing; how-
ever, these often have issues with sourcing (animal-ethical),
reproducibility (batch to batch variation), and tunability (lim-
ited control over mechanical and biochemical signals). The
vast majority of synthetic resins developed for vat photopoly-
merization (DLP and SLA) are based on non-degradable
poly(acrylates). Degradable resins based on poly(lactide),[4]
poly(propylene fumarate),[3b,5] poly(caprolactone),[6] and
poly(glycerol sebacate)[7] have been previously developed,
yet the chemical diversity of available resins remains low.
Current trends in the development of resins for DLP either
focus on softer gel formulations (0.1–100 kPa)[2e,8] or hard
highly-crosslinked resins (100’s of MPa).[9] There is a prohibitive
lack of resins to fabricate scaffolds in the mechanical regime
of elastomeric tissues like cartilage and vessels (low MPa, high
elongation).
Here, we investigated whether poly(ester) urethane acrylate
oligomers could be used to create synthetic DLP resins suitable
for use in tissue engineering. Aliphatic poly(ester)s (here d,l-
lactide, LA, and 𝜖-caprolactone, CL) are ideal candidates for plat-
form materials, due to their wide adoption in the field, excel-
lent biocompatibility, and tolerated hydrolytic degradation prod-
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ucts. (Meth)acrylated poly(ester)s have previously been explored
as biodegradable photoresins,[4,10] and much is known about
the tunability of degradation rates as a function of copolymer
composition.[10b,11] We designed a small library of photopolymer-
izable poly(ester) urethane acrylate polymers, then created photo-
cured scaffolds via DLP, studied the fabricated scaffolds’ physical
properties (mechanical and morphological), tested the cellular re-
sponse on the resins, and the compatibility of the constructs in
vivo (Scheme 1).
The photopolymerizable polymers created within this study
are based on a poly(ester) urethane acrylate (PEUAc, Scheme 1
top) architecture made from poly(dl-lactide-co-𝜖- caprolactone),
L-lysine methyl ester di-isocyanate (LDI), and hydroxylethyl acry-
late (HEA). The use of a biobased and biodegradable LDI can
form a partially segmented polymer architecture before the at-
tachment of an acrylate to chain termini (Scheme 1). This
biobased urethane both serves to increase the mechanical prop-
erties of the resins, and to provide benign degradation products
upon hydrolysis or enzymatic cleavage. Poly(ester) diols for the
production of lysine-based urethanes have become established
as promising biodegradable polymers for a variety of biomedi-
cal applications in the past decades.[12] In order to access softer
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Scheme 2. Synthetic routes to produce PEUAc, 1. Route I, left; A two-step divergent route to produce the final polymer architecture is possible via
sequential addition of the LDI, 3 and the HEA, 5 via intermediate isocyanate terminated polymer 4. Route II, right; Creating the LDI-HEA capping agent
(6) first in a separate reaction enables the synthesis of the PEUAc (1) in a single step from the poly(ester) (2) in a convergent route.
elastomeric materials with higher elongation we have chosen
to not create highly cross-linked resins. Our library explored
PEUAcs in the 1–10 kg mol−1 (Mn ) range.
Scaffolds produced by new resins require thorough biocom-
patibility testing,[13] as well as investigation into their ability to
support cellular function and growth. The surfaces of fabricated
constructs and their chemical composition both influence pro-
tein adsorption and cellular interaction. Our printed PEUAc sur-
faces were characterized via wettability, topography and protein
absorption to provide initial insight on their blood or tissue
biocompatibility.[14] To determine cytocompatibility, we utilized
L929, a prescribed ISO standard fibroblast cell line, to character-
ize cell viability and utilized HDFs to gain further insight into
cellular morphology.[15]
We found that all the PEUAcs synthesized to be amenable
to fabrication via DLP. These polymers represent some of the
highest molecular weights (Mw 20 kg mol−1 ) with low (30 wt%)
reactive diluent modifiers printed for biocompatible polymers.
The fabricated scaffolds possessed mechanical properties in the
low MPa regime, with high elongation at break, and showed
good feature reconstruction. Importantly, the fabricated con-
structs showed high levels of cytocompatibility. We found that
the resins formulated with lower-molecular-weight PEUAcs were
easier to process, due to their lower viscosity; however, the higher-
molecular- weight oligomers created constructs with more prolif-
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eration, higher metabolic activity, and more spread cellular mor-
phologies. All tested resins showed no major inflammatory re-
sponse in vivo. A 4K PEUAc showing a good window of process-
ability and cytocompatibility was scaled up (80 g) to create 3D
printed constructs (gyroid, ear, vascular tree).
2. Results and Discussion
2.1. Polymer Synthesis
The synthesis of the target PEUAcs (1) required a multi-step syn-
thesis that could be approached from either a convergent or a
divergent route (depicted in Scheme 2). Both routes depended
upon the synthesis of 𝛼,𝜔-hydroxy end functionalized poly(ester)
oligomers (2) as a first step. In the divergent approach, 2 was
reacted with LDI (3) to give an isocyanate end functionalized
poly(ester) urethane (4), which was then reacted with an excess of
HEA (5) to afford the PEUAc (Scheme 2, left). Alternatively, in the
convergent approach, the di-isocyanate (3) could be reacted with
HEA (5) in a separate step, in order to form 6, (and byproducts,
vide infra). Then 6 could be used to cap the poly(ester) precur-
sor 2 in a single step (Scheme 2, right). Initially, we sought to
investigate the fidelity of the reaction routes toward the scalable,
controllable, and reproducible synthesis of well-defined resins.
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Table 1. Poly(CL-co-LA) copolymer characterization. small amount of fresh catalyst exhaustively to react with all iso-
cyanates (again followed via IR). After purification of the product
Entry M/Ia) f
PCL
b) Polymer Mn [g mol−1 ] Ðd) by precipitation, 1 H NMR analysis of each step (Figure S6, Sup-
[%] compositionc) porting Information) revealed that the polymer was end-capped
%CL %LA Theory 1 HNMRc) GPCd)
with acrylate groups; however, the ratio between acrylate end-
groups and the ethyl ester methyl protons (from the LDI moiety)
1K P(CL-co-LA) 4/1 79 79 21 1000 1050 3000 1.15 suggested that significant chain extension reactions of LDI with
2.5K P(CL-co-LA) 10/1 79 78 22 2500 2100 4300 1.23 poly(ester) oligomers had taken place. Since our initial results
4K P(CL-co-LA) 16/1 79 77 23 4000 3700 8100 1.28 indicated a lack of suitable control over this sequence, poor pos-
6K P(CL-co-LA) 24/1 79 71 29 6000 6200 13 600 1.37 sibilities for characterization during the reaction sequence, and
10K P(CL-co-LA) 40/1 79 69 31 10 000 9900 20 000 1.28 limited amenability for library formation, we chose to employ the
a) b)
convergent route (vide infra) for the remainder of this study.
Total monomer to initiator molar ratio; Molar fraction of CL in the feed (weight
c)
ratio CL to LA is 75 to 25); Molar ratio estimated by 1H NMR end-group analysis;
d)
GPC with DMF (0.1% LiBr) as eluent and PMMA standards.
2.1.3. Convergent Synthetic Route (Route II)
2.1.1. Synthesis of Poly(ester) Oligomers In the convergent route, the LDI was first reacted with HEA to
prepare compound 6, a bifunctional compound containing both
The poly(ester) (2) oligomer was prepared by ring-opening poly- an isocyanate and acrylate functional group. Utilizing an LDI to
merization of LA and CLusing triethylene glycol as an initiator HEA molar ratio of 1:1 (1:0.5 functional group ratio), a product
and stannous octoate as a catalyst. Poly(ester)s with molecular mixture is expected due to the regioisomeric isocyanates within
weights (Mn ) ranging from 1000 to 10 000 kg mol−1 were targeted LDI coupled with the sub-stoichiometric amount of HEA added
at a CL to LA weight ratio of 75 to 25 (79 to 21 molar ratio, Mn , (Figure 1). Assuming complete reaction, products 6a, 6b, and 6c
determined via 1 H NMR, Table 1). For reference, these polymers are likely to be formed along with unreacted starting material
have on average between 5.0 and 48.9 units polymerized on each LDI. Toward creating well defined polymers in this multi-step re-
side of the triethylene glycol initiator. action, one would like to favor or at least control the formation of
The 1 H NMR spectrum of the poly(ester) shows major sig- 6a and 6b (which would cap the poly(ester)), while minimizing
nals of the LA methine protons at 5.05–5.24 ppm and the CL- the unreacted LDI (leading to chain extension) and 6c. Conve-
CH2 C(O) protons at 2.30–2.43 ppm and allowed the calculation niently, 6a–c can be distinguished by their NH protons chemical
of monomer incorporation and polymer composition (Table 1; shift with the aid of 1 H-15 N 2D NMR (Figure 1a; Figures S7, S8,
Figure S1, Supporting Information). Poly(ester)s of lower molec- Supporting Information). The 1 H NMR spectrum showed dis-
ular weights had higher conversions (associated with the higher tinct signals of the NH protons of 6a at 5.36 ppm (doublet), of 6b
amount of initiator and low viscosity) as well as a polymer compo- at 4.83 ppm (triplet) and of 6c at 5.41 and 4.90 ppm (doublet and
sition closest to the feed ratio (79% PCL in feed). As higher molec- triplet, respectively).
ular weights were targeted, lower conversion of caprolactone led Wanting to determine if the target compounds (6a, 6b) could
to a slight drift in polymer composition (69% PCL in polymer be selectively enriched, investigating the LDI-HEA reaction at
for 10K). The higher amount of lactyl units in the poly(ester) 0, 22, and 40 °C resulted in a constant statistical molar ratio
oligomer was attributed to the well-known higher rate of ring- (6a+6b):6c of 65:35 (Figure 1b). Yet, following the reaction in real
opening polymerization of lactide compared to ɛ-caprolactone.[16] time (at 22 °C) by NMR showed that the isocyanate connected to
A brief kinetic study of the polymerization of the 2.5K P(CL- the secondary carbon in LDI (alpha to the ethyl ester, forming 6a)
co-LA) revealed that this low- molecular-weight oligomer reached is slightly more reactive than the isocyanate on the primary car-
the targeted molecular weight after approximately 3 h via GPC bon (forming 6b) (Figure S9, Supporting Information). Further
and 1 H NMR (Figures S2, S3 and Table S1, Supporting Informa- analysis of the product outcomes as a function of temperature
tion), with slight increases in dispersity moving toward the 24- revealed that the ratio of 6a:6b is indeed affected by the tempera-
h reaction time. Since higher-molecular-weight polymers within ture of the reaction, with higher selectivity at higher temperatures
the library would require more time to fully polymerize (lower (40 °C, Figure 1c). The reasons for this observation in selectiv-
initiator concentration), the entire series was allowed to poly- ity for the different regioisomers, without significantly affecting
merize for 24 h for consistency. Detailed microstructure analy- the unreacted:mono-:di-substituted product profile, remain to be
sis shows a drift from randomness at low molecular weight (R = elucidated.[17]
0.93) to segmented at higher molecular weights (R = 0.37), sug- Since the LDI-HEA mixture prepared at 40 °C resulted in the
gesting considerable trans-esterification in our lower molecular highest enrichment of a single species, this was used to modify
weight oligomers (Figures S4, S5, Supporting Information, for the poly(ester). Analysis of the 1 H NMR of the final polymer (1,
polymer microstructure analysis). PEUAc, Figure 2, 13 C NMR Figure S10, Supporting Information)
revealed the disappearance of the hydroxyl end-groups (2.7 ppm)
2.1.2. Divergent Synthetic Route (Route I) of PEUAc (1) and the appearance of the acrylate protons at 5.86–6.45 ppm and
methyl protons of the ethyl ester at 1.39 ppm. Monitoring the
In the divergent approach, the poly(ester) oligomer was first end- reaction and analysis of the product by IR confirmed that all iso-
group capped with an excess of LDI, following reaction comple- cyanates within the mixture were consumed (Figure S11, Sup-
tion via IR. After 6 h, an excess of HEA was added along with a porting Information). From the ratio of the newly formed end

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Figure 1. Products of the reaction of l-lysine diisocyanate with hydroxyethyl acrylate at a molar ratio of 1:1. a) NH proton chemical shifts from 1 H-15 N
2D NMR confirming the direct coupling of these protons to Nitrogen. b) Percentage of monoacrylate-functionalized isocyanates formed as a function
of temperature. c) Molar ratio of 6a to 6b as a function of temperature.
Figure 2. 1 H NMR spectrum of PEUAc 1 (4K) in CDCl3 .
groups and the poly(ester) backbone signals, a degree of func-
tionalization of 0.63–0.66 was determined for all polymers syn-
thesized, presumably due to some chain extension as supported
via GPC (Table S3, Supporting Information).
The detailed analysis of the two reaction routes showed that the
convergent synthetic approach provided simpler handling, more
chances for the characterization of intermediates, and ultimately
allowed easier scale up to 80 g batch reactions.
2.2. Resin Formulation
In this study, we chose 2-(2-ethoxyethoxy)ethyl acry-
late (EOEOEA) as a biocompatible reactive diluent, and
diphenyl(2,4,5-trimethylbenzoyl)phosphine oxide (TPO) as
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the photo-initiator. EOEOEA, is a well-established and bio-
compatible monofunctional acrylate, used extensively in dental
applications.[18] TPO also has established biocompatibility and
is well-dispersed in organic media/hydrophobic polymers. We
observed that 30 wt% EOEOEA was the minimum amount of
reactive diluted needed to fabricate our highest molecular weight
resins (PEUAc 10K) and was kept constant across the series.
2.2.1. Thermal Characterization
Thermal characterization of the PEUAc polymers via differential
scanning calorimetry (DSC) showed that only the polymer
with molecular weight of 10 kg mol−1 was a semi-crystalline
material, with a melting temperature of approximately 45 °C
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Table 2. Mechanical properties of PEUAc 2D films.
PEUAc 2D films Polymer microstructural magnitudes Young’s modulus [MPa]
l l
CL LL
1K P(CL-co-LA) 5.19 1.36
2.5K P(CL-co-LA) 4.89 1.46
4K P(CL-co-LA) 5.38 1.93
6K P(CL-co-LA) 5.87 2.41
10K P(CL-co-LA) 8.69 3.90
Values are given as mean ± SD from 5 samples.
(Figure S12, Supporting Information). Furthermore, after
crosslinking and the formation of a thermoset, the melting
transition of the 10K PEUAc shifted toward 30 °C.
2.2.2. Mechanical Characterization
In order to characterize the elastomeric nature of the cross-linked
resins, we chose to investigate the mechanical properties of the
resins via tensile testing. (Table 2; Figure S13, Supporting In-
formation). Using standard dog bone samples, the most notice-
able effect of the molecular weight of the polymers on the tensile
properties was elongation at break. The 10K PEUAc shows the
highest elongation at break (586%), with a systematic decrease
to more brittle constructs in the lower molecular-weight resins
(1K PEUAc, 19%). The ultimate tensile strength also generally
increased as the polymer used in the resin’s molecular weight
increased (from 0.5 to 1.8 MPa, approximately that of vessels and
arteries). Of note, in the 10K PEUAc, we noticed the most plas-
tic deformation within the series. On the other hand, the trend
in the moduli of the resin library was bimodal. The 1K PEUAc
resins, having the highest cross-link density, also show the high-
est Young’s modulus (3.10 MPa). Meanwhile, the Young’s mod-
ulus decreased in the 2.5K and 4K PEUAcs (1.95 and 1.38 MPa,
respectively), attributed to their lower crosslink density. However,
the Young’s moduli in the 6K and 10K polymers increased and
is currently attributed to regions of crystallinity in the final con-
struct reinforcing the material (vide supra, supported by DSC
data). We hypothesize that the increase in the block length of CL
segments for the higher molecular weight polymers (6K and 10K)
contributes to crosslinking of the networks formed.
Together, this data shows that the mechanical properties of
the scaffolds can be tailored partially for an envisioned applica-
tion, and the moduli are in the low MPa regime. Interestingly,
this library breaks the general trend in biodegradable polymers[3c]
showing formulations with a similar modulus and an increase in
elongation a break; in this series we observe even a slight increase
in tensile strength with an increase in elongation a break.
2.2.3. Degradation Studies
The degradation and biocompatibility of poly(lactide-co-
caprolactone) copolymers is well established; degradation prod-
ucts include 6-hydroxycaproic acid, lactic acid, and oligomers of
the polymer. The LDI and HEA polymer chain ends will further
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Ultimate tensile strength [MPa] Elongation at break [%]
3.10 ± 0.14 0.52 ± 0.08 19 ± 4
1.95 ± 0.13 1.04 ± 0.06 107 ± 11
1.38 ± 0.13 0.93 ± 0.19 119 ± 28
2.08 ± 0.21 2.70 ± 0.48 351 ± 89
2.90 ± 0.64 1.76 ± 0.38 586 ± 70
degrade into lysine and there will remain the kinetic chain of the
HEA polymerization.[19a] While the aim of the study was not to
confirm these degradation products, we were more interested to
study how the resin formulation and molecular weight affected
the degradation of the resins under hydrolytic conditions. We
first turned toward accelerated degradation conditions (0.1 m
NaOH, at 37 °C) to test our materials; however, all resins de-
graded after 3 days. The rapid degradation rate was not readily
quantifiable using weight loss, as the films and constructs lost
integrity as shown with optical images (Figure S14a, Supporting
Information). Qualitatively, the 1K and 2.5K PEUAc degraded
and fully disintegrated, 4K and 6K degraded into small pieces,
and 10K was able to maintain its integrity.
We then employed slower controlled degradation in water at
room temperature (around 20 °C) in order to capture the degra-
dation with slower kinetics. Shown in Figure S14b, Supporting
Information, all the resins slowly degraded over the course of the
22 days experiment. We observed no clear trends with respect
to molecular weight and degradation. All samples degraded with
around 5–12% mass loss over the experiment, with the 4K PEUAc
sample degrading with a maximum of 20% loss. These exper-
iments show that degradation of these constructs is relatively
rapid, attributed to the incorporation of LA into the resin, and
that the degradation rate is not influenced heavily by the molec-
ular weight of the macromer. In a similar platform, the degrada-
tion kinetics have been shown to be more reliably tuned via the
copolymer composition.[19b]
2.3. Surface Analysis
A biomaterial’s surface constitutes an important cell–material in-
terface and plays an initial key role in the cellular and tissue re-
sponse in vitro and in vivo.[20] In order to evaluate and under-
stand the biocompatibility of our produced biomaterials, we stud-
ied 2D films fabricated via DLP. The surface properties of the
PEUAc constructs were investigated with SEM, water contact an-
gle, and protein adsorption. SEM analysis (Figure S15, Support-
ing Information) of DLP-fabricated films showed smooth sur-
faces for all constructs using PEUAc<10K, while the 10K PEUAc
possessed rougher irregular features. Surface roughness has pre-
viously been correlated with increases in molecular weight[21] and
crystallinity,[22] yet could also be due to phase separation,[23] or
variations in photochemical events.[24]
Contact angle measures the surface energy that controls the
wettability of the surface and is modulated by surface chemistry
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Figure 3. Wettability and protein absorption of DLP-printed 2D films. a) Contact angle measurement of different PEUAc molecular weight displayed a
range of wettability (70–90°). 6K PEUAc is statistically significantly hydrophilic compared to 1K PEUAc film surface. b) BCA assay presented a similar
amount of protein absorbed from all films. Statistical analysis was performed by one-way analysis of variance (ANOVA) with Bonferroni’s multiple
comparison test (*p < 0.05).

Figure 4. Cell attachment, proliferation, and metabolic activity. a) L929 cells adherence (day 1) and proliferation (day 3) on PEUAc films of different
molecular weights (x-axis). b) Normalized metabolic activity (normalized to 10 000 cells) attached to PEUAc films of different molecular weight at day 1
and 3. Black stars and lines display the statistical comparison between day 1 and 3, while red and blue stars and lines illustrate statistical analysis at day
1 and 3, respectively, between the PEUAc films of different molecular weight. Statistical analysis was performed by two-way analysis of variance (ANOVA)
with Bonferroni’s multiple comparison test (*p < 0.05, **p < 0.01, ***p < 0.001). Blue $$$ display the statistically significant value (p < 0.001) of 10K
(DNA assay) and 4K (metabolic activity) to the other PEUAc molecular weights at day 3.

and topography.[25] As samples increased in molecular weight Taken together, these surface analysis results suggest small dif-
from 1K to 6K, we observed decreasing contact angles from 85° to ferences between the surfaces of the fabricated constructs. The
75°, corresponding to increased hydrophilicity (Figure 3a). How- 10K construct does show surface roughness not observed in other
ever, the only statistically significant difference observed was be- films, and the 6K does show a significantly different contact an-
tween 1K and 6K PEUAc films (p < 0.05 by ANOVA). The 10K gle (≈10°); however, all the films show a similar amount of ad-
PEUAc film showed an intermediate mean contact angle of 80°, sorbed protein. These results would forecast small differences in
which could result from its higher surface roughness, as surface the cell-material interactions on different constructs.
roughness is known to increase hydrophobicity.[26]
Protein adsorption is the first step that occurs after in vivo
2.4. In Vitro Studies of Printed Films
implantation or in vitro seeding of a biomaterial and has
been known to be dependent on the biomaterial’s surface
2.4.1. Cell Proliferation and Metabolic Activity
properties.[27] We found similar adsorption from all PEUAc films
regardless of their molecular weight (Figure 3b). Relatedly, Xu
We further tested the biocompatibility of the PEUAc series by
et al.[28] studied the correlation between surface wettability to pro-
seeding them with ISO standard L929 fibroblast cells. Quantifica-
tein adhesion force and discovered a step dependence connec-
tion of DNA revealed significantly lower L929 attachment at day
tion. This may suggest that because of the small wettability range
1 (Figure 4a) on the two lower molecular weight PEUAc films (1K
of the different PEUAc films, the differences in protein adsorp-
and 2.5K), compared to higher molecular weight (4K, 6K, 10K).
tion are negligible.
More striking, cell proliferation (at day 3) was statistically lower

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Figure 5. Cell viability study via live/dead staining. Qualitative analysis on the amount of attached live (green) and dead (red) L929 cells shows live and
dead cells found on the films at day 1 and 3. All attached cells were found to be alive. No dead cells were found. High autofluorescence was observed
from the 10K PEUAc films compared to the other PEUAc films. Scale bars represent 100 μm.
on the 1K and 2.5K films compared to the 4K, 6K, and 10K PEUAc
films (Figure 4a). Higher proliferation rates were systematically
observed as the molecular weight of the construct increased, and
10K PEUAc was observed to have the highest proliferation. Stud-
ies on molecular weight and its effect on cell response and pro-
liferation have been previously performed and are dependent on
polymer and cell type as well as scale of molecular weight.[29–31]
Comparing the metabolic activity of the cells also at day 1 and
3, we observed that the 4K PEUAc films created an increase in
metabolic activity, while the other materials led to a slight de-
crease at day 3 (Figure 4b). Similarly, the total metabolic activity of
seeded L929 was statistically lower on the PEUAc films of lower
molecular weight when compared to films of higher molecular
weight (Figure S16, Supporting Information).
2.4.2. Cell Viability and Cytotoxicity of PEUAc Films
Live/dead staining displayed high amounts of viable cells on
PEUAc films ≥4K and lower amounts on 1K and 2.5K PEUAc
films (Figure 5). Moreover, more elongated and spread cell mor-
phologies were observed in high molecular weight (4K and above)
PEUAc films. Further analysis showed that live cells were found
floating in the lower-molecular-weight samples, in line with the
lower numbers of attached cells shown above (Figure S17, Sup-
porting Information). These data suggest that the films were non-
toxic to cells in spite of their low adhesion. Additional DNA as-
say analysis was conducted to confirm cytocompatibility of 1.5K
and 2K PEUAc, in which films were placed on transwell hover-
ing L929 cells seeded on tissue culture-treated wells (Figure S18,
Supporting Information). No significant difference was seen in
amounts of cells when comparing to the control wells without
films, confirming that the 1.5K and 2K PEUAc films were non-
cytotoxic substrates.
2.4.3. Cell Morphology
In order to investigate the cellular morphology on the PEUAc
resins, we imaged the actin cytoskeleton, which is a structural
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protein network essential for various biological functions.[32,33] To
be able to visualize and generate a well-defined actin cytoskele-
ton analysis, HDFs were used because they showed more pro-
nounced actin filaments (Figure S19, Supporting Information).
At day 1, fewer HDF attached to 1K and 2.5K PEUAc films, and
were also rounder and less spread out, compared to cells found
on the higher molecular weight (4K and above) PEUAc films (Fig-
ure 6). At day 3, the HDF cells on 1K and 2.5K films eventually dis-
played spreading and enhanced actin filaments. In comparison,
HDFs cells found in higher molecular weight PEUAc films at
day 3 were densely populated and spread longitudinally. Though
documented here with two different cell types, the correlation be-
tween cell proliferation and degree of cell spreading has previ-
ously been well documented.[34]
2.5. In Vivo Implantation
With promising cytocompatibility data and the indication of cells
interacting with the materials, we next wanted to investigate if
DLP fabricated objects were implantable and whether they were
tolerated in vivo. For this investigation, we explored both printed
films (2D), and simple gyroid printed scaffolds (3D, vide infra).
We utilized subcutaneous implantation in a pig model, and after
retrieval of the tissue found no indications of a host immune re-
sponse on the short time frame. The scaffolds remained intact,
and the only observable tissue damage was hemorrhage around
the site of implantation (Figure 7). While this is only an initial
test of compatibility in vivo, we find that the materials developed
in this study are suitable for implantation, do not provoke a large
immune response (Masson’s trichrome staining and Picro Sirius
red staining is depicted in Figure S20, Supporting Information),
and are well-situated for further and more in-depth tissue forma-
tion assays in the future.
2.6. 3D Fabrication of Objects
Since the 4K PEUAc resins were observed to possess good cyto-
compatibility, support the adhesion and proliferation of model
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Figure 6. Phalloidin (green) and DAPI (blue) staining to visualize actin filaments and nuclei, respectively, of HDF cells. Cells seeded on 1K and 2.5K
PEUAc films displayed rounded morphology at day 1 and squared-like spreading at day 3. HDF cells seeded on 4K, 6K, and 10K PEUAc films exhibited a
denser population and bilateral spreading. Scale bars represent 50 μm.

Figure 7. Hematoxylin & Eosin (HE) staining on pig skin cryosections. a) 4× magnification on 4K scaffold showing that around the implanted scaffold
there is hemorrhage of the tissue, the scaffold is indicated by the red arrow. b) On a 20× magnification this can be further confirmed as the red blood
cells (blue arrow) are clearly visible. c) 4× magnification on the 1K film showing that around the implanted scaffold there is hemorrhage of the tissue,
the scaffold is indicated by the red arrow (flipped over due to cutting). d) On a 20× magnification this can be further confirmed as the red blood cells
(blue arrow) are clearly visible.

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Figure 8. 4K PEUAc printed into 3D scaffolds of different shapes: a) gyroid structure, b) ear, c) vascular tree. Scale bars represent 5 mm.
fibroblasts, and exhibited lower viscosity (compared to the 6K
and 10K resins), the 4K PEUAc was chosen for scale up and 3D
printing of objects. Using the methods outlined above, we found
good reproducibility and scalability to create this polymer in an
80 g batch reaction, without the need for special equipment. The
DLP printer was slightly modified to reduce the amount of resin
needed for printing 3D objects, as previously reported.[35] A new
printhead and a corresponding resin vat were designed and in-
stalled so that small structures with ≈3 g of resin could be fabri-
cated. Example structures that could be rapidly manufactured in-
clude a 1.5 × 1.5 cm gyroid, an anatomically correct ear, or a sam-
ple of a vascular tree (Figure 8). These samples exhibited good
feature reproduction from the model structures and showed the
ability of the resin to print complex 3D surfaces and overhanging
edges, both hallmark advantages of the DLP printing technique.
More detailed analysis of print fidelity and feature reconstruction
has been reported in some of our previous work.[35]
3. Conclusions
Herein we report the synthesis, characterization, in vitro, and
in vivo testing of PEUAc oligomeric macromonomers used as
resins for DLP of biocompatible and biodegradable tissue engi-
neering constructs. By varying the molecular weight (1K to 10K)
of the macromonomers, we found that there was a trade-off be-
tween fabrication performance and the ability for the fabricated
constructs to support cellular adhesion and growth. Lower molec-
ular weight (1K and 2K) PEUAcs were the easiest to process, yet
the higher molecular weight (≥4K) PEUAcs showed the most
promising cellular behavior. All resins were processable via DLP
and cytocompatibility, yet we find that a 4K PEUAc provided the
best window of processability and cell–material interactions.
Constructs fabricated from these resins provided suitable im-
plantation into an in vivo model, without signs of an early im-
mune response. Furthermore, the mechanical properties of these
resins provide access to previously overlooked stiffness ranges
(low MPa) for DLP materials. This could provide the ability to
create constructs that mimic the tensile moduli of the skin, liver,
kidney, cornea, arteries, and veins.[36] The synthetic methodology
and characterization outlined in this study allow scalability of the
resin synthesis via a well-defined reaction pathway (80 g batches
performed) that can be standardized toward production. These
elastomeric and biocompatible resins will be further explored for
the creation of functional tissue engineering scaffolds.
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4. Experimental Section
Materials: 𝜖-Caprolactone (CL, 99%, VWR, The Netherlands) and
triethylene glycol (TEG, 99%, Sigma- Aldrich, The Netherlands) were
dried over CaH2 and distilled before use. l-lysine ethyl ester di-
isocyanate (LDI, 97%, Sigma-Aldrich, The Netherlands) was stored
over molecular sieves (4 Å) and used without further purification.
d,l-lactide (LA, 97%, VWR, The Netherlands) was recrystallized from
dry toluene. 2-hydroxyethyl acrylate (HEA, 97%), which was dried over
molecular sieves (4 Å), and anhydrous ethanol were provided by
VWR (The Netherlands). Tin(II) 2-ethylhexanoate (stannous octoate,
SnOct2 ), diphenyl(2,4,6-trimethylbenzoyl)-phosphine oxide (TPO, 97%),
anhydrous toluene, and tri-ethylene glycol (TEG) were supplied by Sigma-
Aldrich. 2-(2-Ethoxyethoxy)ethyl acrylate (EOEOEA, 98%, stabilized with
MEHQ, >98.0% (GC)) was obtained from TCI AMERICA.
Synthesis of Poly(𝜖-caprolactone-co-d,l-lactide) (P(CL-co-LA), 2): All
copolymerization were carried out in the bulk at 140 °C under a dry argon
atmosphere for 24 h using triethylene glycol as an initiator and Sn(Oct)2
as a catalyst. The following is an illustrative example for the synthesis of 1K
P(CL-co-LA), with a molecular weight of 1000 and PCL:PLA weight ratio of
75:25 (corresponding to molar ratio of 79:21). In the polymerization, pre-
determined amounts of LA (5.0 g, 34.7 mmol), CL (15.0 g, 131.2 mmol),
and TEG (3.0 g, 19.9 mmol) were simultaneously added to a polymeriza-
tion flask and melted. The flask was purged for 30 min with a dry argon
stream. Then, stannous octoate was added to give a co-monomer: cata-
lyst molar ratio of 2000:1 and stirred for 24 h. The resulting copolymer
was isolated by precipitation in cold ethanol, centrifugation, and vacuum
drying. The desired product was a transparent viscous liquid with a yield
of 22.2 g, 96.3%. 1H NMR (700 MHz, CDCl3, 𝛿, Figure 1a): 5.05-5.24 (q,
–CH–, P-LA), 4.06-4.34 (t, –CH2CH2 O–, PCL and OCOCH2 CH2O–, TEG),
3.62-3.70 (–CH2 O(CH2 )2 OCH2 –, TEG), 2.30-2.43 (t, –OCH2 –, PCL), 1.65
(m, –CH2 CH2 CH2 –, PCL), 1.45-1.60 (d, –CCH3 , PDL- LA), 1.35-1.42 (m,
–CH2 CH2 CH2 –, PCL).
Divergent Synthesis Method to Prepare Poly(𝜖-caprolactone-co-lactide)-
LDI-HEA (PEUAc, 1): In a typical example of the divergent poly(ester)
urethane acrylate preparation, 1K P(CL-co-LA) (10.0 g, 20.0 mmol OH)
was dissolved in 100 mL of dry toluene and added to a solution of LDI
(9.9 g, 40.0 mmol) in 100 mL of toluene under a dry argon atmosphere at
40 °C. Subsequently, stannous octoate was added at a 1% molar ratio of co-
monomers. The reaction was followed by monitoring the decrease of the
NCO vibrational stretch at 2260 cm−1 and the disappearance of the OH
stretch at 3365 cm−1 from 1K P(CL-co-LA) using FT-IR after 6 h. Thereafter,
to the reaction mixture HEA (4.6 g, 40.0 mmol) was added dropwise. The
reaction was performed overnight and followed with FT-IR until the disap-
pearance of the vibrational stretch of the NCO group at 2260 cm−1 . The
polymer was concentrated under reduced pressure and purified by precip-
itation in cold ethanol. Finally, the PEUAc was dried at room temperature
under vacuum until it reached constant weight, yield 32.1 g, 97.5%. 1H
NMR (700 MHz, CDCl3, 𝛿, Figure S2, Supporting Information): 5.89-6.46
(dd, double bonds, HEA) 5.54, 5.39, and 4.85 (d and t, –OCNH–) 5.05-5.24
(q, –CH–, PDL,LA), 4.06-4.34 (t, –CH2 CH2 O–, PCL and –OCOCH2 CH2 O–,
TEG and –OOCCH–, –OCH2 CH3 , LDI and –OCH2 CH2 O–, HEA), 3.62-
3.70 (–CH2 O(CH2 )2 OCH2 –, TEG and –OCNHCH2 CH2 –, LDI), 2.30-2.43
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(t, –OCH2 –, PCL), 1.65 (m, –CH2 CH2 CH2 –, PCL and –CH2 (CH2 )3CH–,
LDI), 1.45-1.60 (d, –CCH3 , PDL,LA), 1.35-1.42 (m, –CH2 CH2 CH2 –, PCL)
and 1.29 (t, –CH2 CH3 ).
Convergent Synthesis Method: In addition, an alternative synthesis
route was investigated to prepare the PEUAc (1). First, LDI was reacted
with HEA followed by the reaction of the obtained “capping mixture” with
the poly(ester) diol pre-polymers (P(CL-co-LA)) as described below.
𝛼-Isocyanate-𝜔-Acrylate-Carbamate (6a and 6b): Hydroxyethyl acrylate
(HEA, 465 mg, 4.0 mmol) was added dropwise to a solution of l-lysine di-
isocyanate (LDI, 905 mg, 4.0 mmol) containing stannous octoate (6 mg, 1
mol%) in 5 mL of toluene. Reactions were performed at different tempera-
tures (0, 25, and 40 °C) to determine the influence on the final composition
of the reaction mixture. The mixture was used in the following step without
further purification.
Poly(ester) Urethane Acrylates (PEUAc, 1): In a typical example, 1K
P(CL-co-LA) (10.0 g, 20.0 mmol OH) was dissolved in anhydrous toluene
(100 mL) and added to a solution of 𝛼- isocyanate-𝜔-acrylate-carbamate
mixture (11.0 g, 30.3 mmol isocyanate groups) in 110 mL of dry toluene at
room temperature under a dry argon atmosphere. Subsequently, stannous
octoate was added using a 1% molar ratio of co-monomers. The reaction
temperature was gradually increased up to 40 °C. Progress of the reac-
tion was followed by FT-IR spectroscopy using the vibrational stretch of
the NCO group at 2260 cm−1 . The acrylate end-functionalized poly(ester)
urethane was first concentrated under reduced pressure and subsequently
precipitated into an excess of ethanol in order to remove the catalyst impu-
rities, monomers and side products of the formed 𝜔-acrylate-carbamate-
isocyanate-𝜔-acrylate-carbamate. Finally, the PEUAc-II was dried at room
temperature under vacuum until it reached constant weight, yield 31.8 g,
96.4%. 1 H NMR (700 MHz, CDCl3, 𝛿, Figure 4): 5.89-6.46 (dd, dou-
ble bonds, HEA) 5.54, 5.39, and 4.85 (d and t, –OCNH–) 5.05-5.24 (q,
–CH–, PDL,LA), 4.06-4.34 (t, –CH2 CH2 O–, PCL and –OCOCH2 CH2 O–,
TEG and –OOCCH–, –OCH2 CH3 , LDI and –OCH2 CH2 O–, HEA), 3.62-
3.70 (–CH2 O(CH2 )2 OCH2 –, TEG and –OCNHCH2 CH2 –, LDI), 2.30-2.43
(t, –OCH2 –, PCL), 1.65 (m, –CH2 CH2 CH2 –, PCL and –CH2 (CH2 )3CH–,
LDI), 1.45-1.60 (d, –CCH3 , P(DL,LA)), 1.35-1.42 (m, –CH2 CH2 CH2 –, PCL)
and 1.29 (t, –CH2 CH3 ).
Scaffold Fabrication: 2D Film Printing and Sterilization: PEUAc of all
molecular weights were mixed with 30% EOEOEA and 1 wt% TPO and cast
onto glass slides. Circular 2D films of 9.5 mm in diameter and 0.5 mm in
thickness were printed with the commercially available DLP printer (ARM
10, Roland). The layered films were washed with acetone to remove any
uncured resin, and then left overnight in a vacuum oven to dry. Next, films
were moved into 48-well plates and secured with o-rings (Eriks BV). Ster-
ilization was performed by UV exposure (365 ± 5 nm, 10 mW cm−2 ) for
30 min.
Scaffold Fabrication: 3D DLP Printing of Gyroids: 3D scaffolds were fab-
ricated with a commercially available DLP printer (ARM 10, Roland) with a
customized printing vat and a newly designed heating stage that allowed
printing at temperatures up to 80 °C. The synthesized resin was preheated
to 50 °C and mixed with 30 wt% of EOEOEA and 1 wt% of TPO. After
thoroughly mixing, the resin was poured into the preheated vat and the
designed scaffold was fabricated at temperatures of around 55 °C.
Nuclear Magnetic Resonance Spectroscopy: 1 H, 13 C, and 15 N-correlated
NMR spectra were recorded on a Bruker Avance III HD 700-MHz spec-
trometer equipped with a cryogenically cooled three-channel TCI probe.
NMR samples were prepared in 5 mm NMR tubes with deuterated chlo-
roform (CDCl3) or toluene-d6 as solvents. TMS was used as an internal
standard for calibration of the 1 H and 13 C chemical shifts. 15 N chemi-
cal shifts were externally referenced to the single 15 N resonance in neat
nitromethane-d3. Two types of 2D NMR sequences were assayed in this
study: a heteronuclear single-quantum correlation 15 N-1 H HSQC ver-
sion and a heteronuclear multiple bond correlation 13 C-1 H HMBC, both
recorded at natural abundance. In order to determine the reactivity of
the different isocyanate groups in the reaction of LDI with HEA to 𝛼-
isocyanate-𝜔-acrylate-carbamate, the reaction in deuterated toluene was
followed in real-time by 1 H NMR at three different temperatures (0, 22,
and 40 °C), and conversion rates were analyzed as a function of reaction
time.
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Molecular Weight Measurements: Gel permeation chromatography
(GPC) with N,N-dimethylformamide (DMF) containing 0.1 wt% LiBr as
eluent was performed for all polymers with sample concentrations of 2 g
L−1 . The Shimadzu Prominence-i system comprised an autosampler, a
Shodex KD-G 4A guard column (4.6 × 10 mm) with 8-μm beads, fol-
lowed by a Shodex KD-802 (5 μm, 8 × 300 mm) and a KD-804 (7 μm, 8
× 300 mm) columns, a refractive index detector and a photodiode array
detector at 50 °C with a flow rate of 1 mL min−1 . The GPC system was
calibrated against linear poly(methyl methacrylate) standards with molec-
ular weights ranging from Mp = 600 to 265 300 Da. The samples were fil-
tered through polytetrafluorethylene (PTFE) membranes with a pore size
of 0.2 μm prior to injection.
Thermal Properties: Thermal properties of synthesized polymers were
determined using differential scanning calorimetry (DSC 250, TA Instru-
ments). The measurements were carried out with heat/cool/heat cycles in
the −40 to 200 °C range at heating and cooling rates of 10 °C min−1 and a
nitrogen flow rate of 25 mL min−1 . Universal Analysis software was used
for thermal data analysis, such as the determination of melting and glass
transition temperatures.
Fourier Transform Infrared Spectrometry (FT-IR): A Thermo Scientific
spectrophotometer (Nicolet iS50 FTIR) with Omnic software was used for
data acquisition and analysis. The diamond ATR stage was utilized for all
measurements.
Mechanical Analysis: Tensile tests were carried out on dumbbell-
shaped specimens (n = 5 for each molecular weight) with a standard JIS-
K6251-7 size (10 mm in gauge length (L0 ) × 2.5 mm in width (w)) using an
ElectroForce mechanical tester from TA Instruments. Films with thickness
of 300 ± 15 μm were formed by casting the PEUAc solution at a concentra-
tion of 70% (wt) in EOEOEA as a reactive diluent and 1% (wt) of TPO as
a photointiator in a homemade PDMS mold and photopolymerized under
UV (365 ± 5 nm) for 5 min on each side. Samples were dried under vac-
uum at room temperature for 24 h. Tests were performed at a stretching
rate of 0.1 s−1 . The nominal stress 𝜎 was estimated from the force divided
by the cross-sectional area of the un-deformed sample. The strain 𝜖 was
determined from the clamp displacement divided by L0 . Young’s modu-
lus was calculated by stress divided by strain (E = 𝜎/𝜖) in the low strain
area (<1%). All measurements were performed at room temperature and
repeated at least 5 times.
Surface Topography: Scanning electron microscopy (SEM) was used
to analyze the surface topography of the printed 2D films (n = 3 for each
molecular weight). Films were gold sputtered (Cressington 108 auto) at
40 mA and 100 mTorr for 30 s. Surface topography was observed using an
SEM (XL 30 ESEM-FEG, Philips/FEI).
Wettability: Drop shape analysis on sessile drops was done using
Drop Shape Analyzer (DSA25, Krüss GmbH). Water contact angle mea-
surement of 1 μL in volume was dropped on top of PEUAc films (n = 3 for
each molecular weight) and measured using Drop Shape Analysis software
(DS4, Krüss GmbH).
Protein Absorption: Films (n = 3 for each molecular weight) were
submerged in 500 μL DMEM containing 10% fetal bovine serum (FBS)
and incubated at 37 °C overnight. The biochemical assay based on the
bicinchoninic acid assay (BCA protein assay, Thermo Fisher Scientific,
USA) was used following manufacturer instructions to determine the
total amount of protein adsorbed on the PEUAc film surfaces. After
incubation, films were dried and transferred to 1.5- mL Eppendorf tubes
followed by the addition of the bicinchoninic acid working reagent
(Thermo Fisher Scientific, USA) and incubated for 30 min at 37 °C. The
absorbance was read on a CLARIOstar plate reader (BMG LABTECH) at
562 nm.
Degradation: Accelerated degradation of polymer was evaluated us-
ing procedure as previously reported under basic conditions using 0.1 m
sodium hydroxide solution. Briefly, a dog bone shape sample was placed
in a glass vial, and 10 mL of the solution was added to the glass vial. The
glass vial was placed in water bath set to 37 °C. After 72 h, the samples were
removed from the water bath for recording wet weight. Since polymer al-
ready degraded after 3 days under accelerated degradation conditions and
weighing was not possible, the degradation was documented with images
(Figure S14, Supporting Information).
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Then, degradation was investigated with polymers in water at 20 °C (lab
temperature). Dog bone shape samples (in triplicate) were used for degra-
dation study. A sample was placed in an Eppendorf and 2 mL Milli Q water
was added to the Eppendorf to immerse the sample fully in water. Sam-
ples were left at room temperature for degradation. At each time point,
the sample was removed from Eppendorf, dried on a piece of tissue, and
then the weight recorded. Weight loss at each time point was determined
by comparing to the initial weight of each sample.
In Vitro Study—Cell Expansion: L929 fibroblast cell line (NCTC clone
929 [L cell, L-929, derivative of Strain L] (ATCC CCL-1)) and human der-
mal fibroblast (HDF, Cell Applications) were cultured in culture medium
comprising DMEM (Gibco) with Glutamax, 10% FBS (Lonza), and peni-
cillin (100 U mL−1 ) and streptomycin (100 mg mL−1 , Gibco). L929 and
HDF were expanded at initial seeding density of 5000 cells cm−2 and 3000
cells cm−2 , respectively, in culture medium at 5% CO2 humid atmosphere
and 37 °C. For both cell types, cultures were refreshed every 2–3 days and
harvested at 80–90% confluence, for cell seeding.
In Vitro Study—PEUAc Films Cell Seeding: After sterilization, PEUAc
films were washed twice with phosphate-buffered saline (PBS) and incu-
bated overnight in culture medium at 37 °C. For direct seeding to analyze
attachment, proliferation, metabolic activity, and viability, L929 cells were
seeded at 10 000 cells per PEUAc film of different molecular weights. To
study cell morphology, HDF cells were seeded at 5000 cells per film. For
cytotoxicity test, PEUAc films were placed on transwells (8 μm polycarbon-
ate pore size, Corning) on top of cell culture treated wells seeded with L929
cells.
In Vitro Study—L929 Metabolic Activity: Films (n = 3 for each molecu-
lar weight) were gently washed with PBS and incubated with preheated
250 μL of 1:10 PrestoBlue Cell Viability Reagent (Thermo Fisher Scien-
tific) in culture medium for 1 h at 37 °C in the dark. Duplicates of 100 μL
solution from each sample were measured with a spectrofluorometer at
fluorescence excitation wavelength at 570 nm and emission at 585 nm
(CLARIOstar plate reader, BMG LABTECH). Fluorescence levels were nor-
malized to the total number of cells to achieve a per-cell metabolic activity
measurement.
In Vitro Study—L929 Adhesion, Proliferation, and Cytotoxicity: The
same films used for Presto Blue were used for DNA analysis to evaluate cell
adhesion and proliferation. Films (n = 3 for each molecular weight) were
washed twice with sterile PBS and stored in an Eppendorf tube at −20 °C
until further processing. Films were then digested at 56 °C for 16 h in a
Proteinase K buffer (1 mg mL−1 Proteinase K, 18.5 μg mL−1 pepstatin A
and 1 μg mL−1 iodoacetamide in Tris-EDTA (Sigma-Aldrich)). DNA quan-
tification was performed with the CyQuant DNA assay (Molecular Probes,
USA) following the manufacturer’s guidelines using a spectrofluorometer
at 480/520 nm excitation/emission wavelength. For cell cytotoxicity analy-
sis (PEUAc on transwell), L929 seeded on tissue culture treated well plates
(n = 3 for each molecular weight) were lysed with Proteinase K buffer
and collected into Eppendorf tubes. All remaining steps to measure the
amount of viable cells followed the same procedure as the film above.
DNA amounts were normalized to a control well plate without films and
transwells.
In Vitro Study—Cell Viability: Live/dead staining was used to evaluate
cell viability. Films (n = 2 for each molecular weight) were washed with
PBS and incubated with 6 μm ethidium homodimer and 4 mm calcein in
Hank’s Balanced Salt Solution (HBSS) at 37 °C for 30 min in the dark.
Images of green live and red dead cells were acquired by the Nikon TI-S
inverted fluorescence microscope.
In Vitro Study—Cell Morphology: Phalloidin and DAPI staining, and
SEM imaging were used to analyze cell morphology. Films (n = 4 for each
molecular weight) were washed with PBS and fixed with 4% paraformalde-
hyde for 15 min at room temperature. Fixed films (n = 2) were then per-
meabilized with 0.1% Triton X-100 for 5 min and blocked with 1% bovine
serum albumin (BSA) for 1 h at room temperature. Cells were then stained
with Alexa Fluor 488 Phalloidin (Thermo Fisher Scientific, 50 μg mL−1 )
for 30 min and DAPI (Thermofisher Scientific, 0.1 μg/ml) for 15 min with
three washing steps in between. Images were taken using a fluorescence
microscope (Inverted fluorescence microscope Nikon TI-S) at 20× magni-
fication. Remaining fixed films (n = 2), after rinsing with PBS, underwent
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dehydration steps of 25-50-70-85-95-100% ethanol, 15 min per step. After
dehydration, films were dried with hexamethyldisilazane (HMDS, Sigma-
Aldrich). Films were then gold-sputtered (Cressington 108 auto) at 40 mA
and 100 mTorr for 30 s, and cell morphology was observed using a Philips
XL30 aESEM-FEG SEM.
In Vivo Study—Animal Study: Three female Dutch landrace pigs, 35–
50 kg, were used in compliance with the Dutch regulations of experiments
on animals. All procedures on animals were approved by the local ani-
mal ethical committee under project license no. AVD1070020174166 and
conducted at the central animal facilities of Maastricht University. If ani-
mals could survive for 8 h after the implantation, they met the inclusion
criteria. No animals were lost during the anesthesia. A total of 12 subcu-
taneous “pockets” were distributed to three animals with one pocket per
limb. Each pocket received a sterilized implant (scaffolds, n = 4; 4K films,
n = 3; 1K films, n = 3; 10K films, n = 2). All animals received premedica-
tion of azaperone 3 mg kg−1 , ketamine 10 mg kg−1 , and atropine 0.05 mg
kg−1 via intramuscular injection. Anesthesia was induced with intravenous
injection of thiopental 10–15 mg kg−1 and maintained by isoflurane and
oxygen after intubation. After fur shaving and disinfection, a 1 cm inci-
sion was made on the limb skin. A subcutaneous pocket around 1 cm ×
1 cm was created by blunt dissection. An implant was placed with its sur-
face touching the fascia layer of the limb without any suture fixation. The
skin incision was closed intracutaneously with Monocryl 4-0 (Ethicon Inc,
Johnson&Johnson, Somerville, NJ). Afterward, laparoscopic surgical pro-
cedures were performed intraperitoneally under anesthesia, as described
in earlier studies.[37] All animals were sacrificed eight hours after the im-
plantation. The pocket tissues containing the implant were collected im-
mediately for histology.
In Vivo Study—Histology: Pig skin tissues were embedded in Opti-
mal Cutting Temperature (OCT) compound (CellPath KMA-0100-00A) and
were cryosectioned at 8 μm. Sections were stained with hematoxylin and
eosin (HE), Picro Sirius red and Masson’s trichrome to determine gen-
eral histochemical characteristics. In brief, frozen sections were thawed to
room temperature and fixed with 4% paraformaldehyde in binding buffer
for 30 min. After fixation, tissue sections were washed twice in demi water.
For HE staining, sections were stained in Hematoxylin solution (Klinipath
4085–9005) for 2 min and developed for 5 min under tap water. Next, sec-
tions were stained in Eosin solution (Klinipath 640380) for 5 min. For Picro
Sirius red, sections were stained as follows: 0.2% phosphomolybdic acid
solution (Sigma HT153) for 5 min, 0.1% Sirius Red solution for 90 min,
and 0.01 m hydrochloric acid for 5 min. For Masson’s trichrome, sections
were stained as follows: Bouin’s solution (Sigma HT10132-1l) 15 min at
56 °C, Hematoxylin for 5 min, Biedrich Scarlet-acid fuchsin for 5 min, Mor-
dant solution for 7.5 min, Alinine blue solution for 5 min, and 1% acetic
acid for 2 min. After all histochemical stainings, sections were dehydrated
and covered in Entallan (VWR1.079.601. 500). Images were taken with a
Leica Application Suite X microscope.
Statistics Analysis: All data graphs are expressed as mean ± s.d. Bio-
chemical assays were performed with triplicate biological sample, if not
stated otherwise. Statistical analysis was done by two-way analysis of vari-
ance (ANOVA) with Bonferroni’s multiple comparison test (p < 0.05), un-
less otherwise indicated in the figure legends. For all graphs the following
applies: * = p < 0.05, ** = p < 0.01, *** = p < 0.001.
Supporting Information
Supporting Information is available from the Wiley Online Library or from
the author.
Conflict of Interest
The authors disclose that one of their co-authors A.D. (DSM Biomedical) is
co-inventor on a patent (EP 2089438B, US 9458256) related to the creation
of urethanes with acrylate functionalities.
© 2023 The Authors. Advanced Healthcare Materials published by Wiley-VCH GmbH

www.advancedsciencenews.com
Data Availability Statement
The data that support the findings of this study are available from the cor-
responding author upon reasonable request.
Keywords
biofabrication, DLP printing, poly(ester), polymer synthesis
Received: December 5, 2022
Revised: February 7, 2023
Published online:
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